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Protein phosphorylation is catalyzed by kinases to regulate a large variety of cellular activities, including growth and signal transduction. Methods to identify kinase substrates are crucial to fully understand phosphorylation‐mediated cellular events and disease states. Here, we report a set of protocols to identify substrates of a target kinase using Kinase‐catalyzed Biotinylation with Inactivated Lysates for Discovery of Substrates (K‐BILDS). As described in these protocols, K‐BILDS involves inactivation of endogenous kinases in lysates, followed by addition of an active exogenous kinase and the γ‐phosphate‐modified ATP analog ATP‐biotin for kinase‐catalyzed biotinylation of cellular substrates. Avidin enrichment isolates biotinylated substrates of the active kinase, which can be monitored by western blot. Substrates of the target kinase can also be discovered using mass spectrometry analysis. Key advantages of K‐BILDS include compatibility with any lysate, tissue homogenate, or complex mixture of biological relevance and any active kinase of interest. K‐BILDS is a versatile method for studying or discovering substrates of a kinase of interest to characterize biological pathways thoroughly.