Mass spectrometry (MS) offers robust, label-free approaches for characterizing ligand-protein interactions through two main strategies: affinity-based and stability-based assays. Affinity-based methods, such as affinity-selection MS (AS-MS) and mass spectrometry binding assays (MSBA), detect ligand-target interactions by identifying bound ligands or measuring displacement of a reporter ligand. Stability-based techniques, including thermal proteome profiling (TPP) and limited proteolysis-MS (LiP-MS), infer interactions based on changes in a protein’s thermal or proteolytic stability. These MS-based workflows enable proteome-wide analyses with high specificity and throughput. However, their application to membrane proteins (MPs)—a major class of drug targets—has been limited by challenges such as structural complexity, low native expression, and poor compatibility with detergent-based MS protocols. Recent advances in membrane mimetic (MM) systems, including nanodiscs, Peptidiscs, and styrene-maleic acid (SMA) polymers, help address these barriers by maintaining native-like lipid environments and preserving functional MP conformations. These mimetics facilitate proteome-scale solubilization of MPs in forms compatible with MS screening. This review outlines key affinity- and stability-based MS approaches and examines their adaptation for MPs. It also highlights how combining MS techniques with MM systems is expanding the reach of high-resolution, functional analysis of MP–ligand interactions.