Activity-based protein profiling (ABPP) has been widely used for proteome-wide cysteine profiling to study functional cysteines in biological systems. ABPP can also be performed in a competitive manner for lead compound discovery and target identification of the lead compound. Recently, we reported a new class of acrylamide-based cysteine probe, NAIA, which has superior cysteine reaction kinetics to capture more functional cysteines. We further established the workflow of using NAIA for cysteine profiling by mass spectrometry (MS)-based ABPP. Here, we describe a step-by-step protocol using NAIA in the MS-based ABPP experiments for profiling functional cysteines in cancer cells/cell lysates and protein target identification of covalent ligands.