Adeno-associated virus (AAV) has become a popular vector for therapeu9c gene delivery . Twelve AAV serotypes which are well characterized in terms of 9ssue tropism and transduc9on efficiency, allow for directed administra9on into target 9ssue . AAVs are composed of a capsid, which is assembled through mul9ple copies of three viral capsid proteins (VP1, 2 and 3) and equipped with single stranded DNA as transgene, which is meant for delivery into pa9ent cells . During produc9on, impuri9es can emerge which lack the desired therapeu9c proper9es. A common impurity which can account for more than 98% of the total of par9cles produced are capsids that have failed to package the gene of interest, rendering them therapeu9cally incompetent . While in some scenarios these by-products can aWract an9-AAV an9bodies, thus ac9ng as decoys, their presence inevitably necessitates the administra9on of higher doses of prepara9ons to achieve a desired therapeu9c effect, fueling the risk of dose-dependent immunogenic and cytotoxic responses . In consequence, AAVs require characteriza9on aeer produc9on and before pa9ent treatment to allow for evalua9on and adjustment of the final par9cle composi9on. Despite being 9me consuming and requiring rela9vely large amounts of material, analy9cal ultracentrifuga9on (AUC) is s9ll considered the gold standard for the characteriza9on of the AAV par9cle composi9on . An alterna9ve approach that has gained aWen9on in recent years is anion exchange chromatography (AEX). The basis for separa9on in AEX is a difference in the isoelectric point of full and empty capsids of reportedly 0.4 units governed by the presence or absence of DNA . This difference causes stronger reten9on of the more nega9vely charged full species on the AEX phase and later elu9on. Commonly, AEX is performed with a gradient of increasing ionic strength and oeen non-linear gradients containing isocra9c holds to achieve elu9on and separa9on . While the isoelectric points of different AAV serotypes are in a similar region of around 6.3, they tend to behave differently on AEX separa9on phases, usually complica9ng the development of generically applicable methods and gradients 11 . Another boWleneck is the inability to differen9ate other impuri9es such as par9ally filled par9cles . The successful analysis of these forms was previously described through AUC, mass photometry and charge-detec9on mass spectrometry (CDMS) . In this contribu9on, we aimed to develop an AEX-based separa9on strategy that can overcome the above discussed limita9ons, i.e., being generically applicable and mass spectrometry friendly. AEX chromatography was performed using a non-porous 3 µm par9cle column with highly monodisperse packing proper9es, facilita9ng the separa9on of large biomolecules with minimal Eddydiffusion and good mass transfer, which generated sharp peaks, high chromatographic resolu9on, and high sensi9vity. Ion exchange chromatography can either rely on salt-gradient elu9on, pH-gradient elu9on, or a mixture thereof. Recent literature demonstrates that either mode can facilitate highly efficient separa9ons of proteins and their isoforms . While salt gradient elu9on requires the use of a high ionic strength mobile phase, pH-gradient elu9on does not, which is clearly favorable when subsequent mass spectrometric interfacing is desired . It was also shown that MS-friendly mobile phases of low ionic strength can outperform conven9onal non-vola9le salt or pH-gradient separa9on systems in terms of separa9on performance, hence, in this study we further expanded our use of pHgradient elu9on for complex biomolecule separa9on .