Lipid systems biology, i.e. the identification of lipid molecular profiles, their dynamics and associated biological pathways, requires the acquisition of complex datasets from different experimental sources. Lipidomic analysis has become a rapidly growing field amongst -omics sciences and a major challenge for analytical chemistry per se owing to the structural diversity of the lipidome. Due to a wide dynamic range of concentrations spanning from pM to μM 1 , the analysis is predominantly conducted with electrospray ionization mass spectrometry (ESI-MS) methods 2 . Although discovery approaches put an emphasis on lipid alteration patterns in a systemic context, description and thorough structural characterization of the highest possible number of molecular species remains an important task. Therefore, liquid chromatography (LC) is coupled with MS detection to facilitate the separation of isomers and decrease the strong ion suppression originating from the multitude of coeluting compounds . To date, most LC/ESI-MS experiments in discovery lipidomics used analytical-bore column dimension with 2.1 mm internal diameter (ID) and up to 150 mm length at 200-500 μL/min flow rates. Reversed-phase (RP) separation using fully or superficially porous silica functionalized with hydrophobic ligands (C4 to C30) remains a method of choice due to the well-studied separation mechanisms and overall robustness of the technique . Recently, several groups demonstrated that nanoscale LC coupling with nanoelectrospray ionization (nLC/NSI-MS), widely utilized in protein biomarker research, may also be a valuable analytical configuration for lipidomic studies. It is especially suitable for deep screening of biological lipid extracts due to benefits of nLC/NSI-MS, such as reduced chromatographic dilution, improved ionization efficiency, low sample and solvent consumption and broader coverage of the lipidome . However, nLC suffers from practical operational problems in form of hardly detectable leaks, large influence of system volumes and associated longer runtimes. In addition, well-trained and skilled scientists are needed to operate this costly nanoflow equipment. Therefore, the bioanalytical community is in general still scared away from implementing nLC-based methods in routine workflows. Microflow LC (μF) using flow rates of 1-50 μL/min appears as an excellent strategy to reduce the detrimental effects of system volumes, increase sample loading and overall system robustness while achieving throughput levels comparable to conventional analytical-bore methods. As a drawback, the associated ionization efficiency and ion sampling improvements are significantly lower in µF compared to nLC/NSI, making it harder to justify the transition . Thanks to new microfabricated monolithic multinozzle (M3) electrospray emitters, it becomes possible to effectively obtain NSI from μF separation via at-source splitting of the incoming effluent . In this work, we evaluate how this μF/mnESI set-up enabled by M3 emitters could be considered for discovery lipidomics. Its capacity to achieve deep lipidome profiling of biological samples in a timeframe comparable to classic LC/ESI-MS measurements is next demonstrated in a proof-of-concept application involving a comparative analysis of lipid extracts from 3D clear cell renal cell carcinoma (ccRCC) cells cultured in 3-dimensional conditions (3D) and exposed to an optimized proprietary multidrug combination therapy 12 .