Membrane proteins represent the majority of clinical drug targets and are actively involved in a range of cellular processes. However, the complexity of membrane mimetics for membrane protein solubilization poses challenges for native mass spectrometry (MS) analyses. The most common approach for native MS analyses of membrane proteins remains offline buffer exchange into native MS-compatible buffers prior to manual sample loading into static nano-ESI emitters. This laborious process requires relatively high sample consumption and optimization for the individual proteins. Here, we developed online buffer exchange coupled to native mass spectrometry (OBE-nMS) for analyzing membrane proteins in different membrane mimetics, including detergent micelles and nanodiscs. Detergent screening for OBE-nMS reveals that mobile phases containing ammonium acetate with lauryl-dimethylamine oxide are most universal for characterizing both bacterial and mammalian membrane proteins in detergent. Membrane proteins in nanodiscs simply require ammonium acetate as the mobile phase. To preserve the intact nanodiscs, a novel switching electrospray approach was used to capture the high-flow separation on the column with a low-flow injection to MS. Rapid OBE-nMS completes each membrane protein measurement within minutes and thus enables higher-throughput assessment of membrane protein integrity prior to its structural elucidation.