The recent success of AAV-based gene therapy applications has created a need for effective analytical tools with high separation efficiency and sensitivity for quality control during rAAV production . Currently, there are seven FDA/EMA-approved biologics using AAV technology to treat a diverse number of diseases and many more in Phase II/III clinical trials . AAV serotype 8 (AAV8) exemplifies the potential of rAAV biologics, demonstrating robust gene transduction and delivery to the liver in preclinical models, including primates and rodents. This serotype has gained considerable attention due to its relatively low immunogenicity. Numerous AAV8-based therapies are currently in clinical trials to deliver genes for hemoglobinopathies and other clinical disorders . Furthermore, studies have shown that AAV8 offers superior transduction and expression in the liver than other serotypes (e.g., AAV2) . The AAV virus consists of a single-stranded DNA genome of ~ 4.7 kb enclosed by an icosahedral capsid shell comprising three viral proteins, VP1, VP2, and VP3, at an approximately 1:1:10 ratio . A significant challenge in AAV gene therapy is the high vector doses needed to produce a therapeutic effect, partly due to the presence of empty capsids . Therefore, it is crucial to minimize empty capsids and other contaminants, as well as to implement a reliable and reproducible method for their detection. Doing so will enhance rAAV production and maximize its efficacy. In addition, AAV particles can aggregate under thermal stress or at high concentrations during manufacturing, storage, and use. For example, it is problematic to administer small volumes of concentrated rAAV vectors to certain sites (e.g., central nervous system) because increasing the concentration of AAV products can lead to aggregation. Indeed, rAAV aggregation is a significant concern. The aggregation of AAV particles can lead to inconsistencies in AAV content, losses during purification, increased immunogenicity, and therapeutic inefficacy . Moreover, VP proteins can undergo truncations, which have been associated with reduced transduction efficacy . The AAV capsid content is one of the most critical quality attributes. rAAVbased therapies are dosed according to the VG titer. Indeed, determining the content of rAAV-based products represents a significant analytical challenge due to their aggregation propensity, high molecular weights, heterogeneity, limited sample amounts, and structural complexity . Common approaches to solve these limitations and to measure multiple AAV attributes include size exclusion chromatography (SEC) or ion-exchange chromatography (IEX) coupled to light scattering detectors or mass spectrometers . Although SEC is an attractive chromatographic strategy for monitoring aggregation, this technique is limited by its separation mechanism, which relies on differences in size in solution to achieve separation. IEX delivers high-resolution separations, but high-salt concentrations might affect the integrity of AAV assemblies . Furthermore, this new generation of biotherapeutics is much larger and more complex than previous antibody-based products. Native mass spectrometry (native MS) has been used for fast AAV analysis 24, 25 , but limitations associated with unresolved peaks in the m/z spectrum preclude the assignment of charge states . On the other hand, charge-detection mass-spectrometry (CD-MS) enables sensitive analysis of heterogeneous AAV assemblies . This single-particle technique allows the determination of each ion's mass by measuring (simultaneously) the mass-charge ratio and charge of individual ions . Thus, empty and filled ratios can be accurately quantified. Mass-photometry (MP) has been used for the analysis of AAV capsids 29, 30 . This single-molecule technique offers high resolution, speed, and sensitivity. Recently, Ebberink et al. compared MP and CD-MS techniques for their ability to characterize AAV capsids . In this study, both techniques were considered valuable tools for characterizing AAV capsids. While MP was highlighted as a simpler and faster technique, CD-MS was deemed to provide superior mass accuracy. Interestingly, the Marty and Jarrold groups have demonstrated the online coupling between SEC and CD-MS for the analysis of large proteins and virus-like particles . An automated CD-MS microfluidic platform has been reported for the analysis of megadalton biotherapeutics . In particular, automated workflows are needed for fast and reproducible assessment of key AAV quality attributes (e.g., capsid aggregation, genome packing, molecular weight, and content ratio). AAV-based products' success relies on the AAV vectors' homogeneity and purity. For quality assurance and safety purposes, it is critically important to evaluate the content of AAV products by separating AAV from its aggregates and other impurities. Protein-based drugs can aggregate during development, manufacturing, or storage, leading to their toxicity and efficacy reduction . This critical quality attribute must be monitored. Offline native (nSEC) has recently been used to fractionate small-sized protein assemblies from breast cancer cells . Although SEC is well-suited for separating and quantifying large biomolecules, it is unable to separate empty and full capsids because they have the same size in solution . Here, we demonstrate that offline nSEC in conjunction with multi-angle light scattering (MALS) and diode-array detector (DAD) is an attractive option to identify and distinguish AAV from its aggregates prior to CD-MS analysis.