Kinases catalyze protein phosphorylation to regulate cell signaling events. However， identifying kinase substrates is challenging due to the often low abundance and dynamic nature of protein phosphorylation. Development of novel techniques to identify kinase substrates is necessary. Here， we report kinase-catalyzed biotinylation with inactivated lysates for discovery of substrates (K-BILDS) as a tool to identify direct substrates of a kinase. As a proof of concept， K-BILDS was applied to cAMP-dependent protein kinase?A (PKA) with HeLa cell lysates. Subsequent enrichment and MS/MS analysis identified 279 candidate PKA substrates， including 56 previously known PKA substrates. Of the candidate substrates， nuclear autoantigenic sperm protein (NASP)， BCL2-associated athanogene?3 (BAG3)， and 14-3-3 protein Tau (YWHAQ) were validated as novel PKA substrates. K-BILDS provides a valuable tool to identify direct substrates of any protein kinase.