The effects of compound loading on the identification of protein kinases (PKs) was examined using two previously reported sepharose-supported PK inhibitors (PKIs): bisindolylmaleimide X (S1) and CZC8004 (S2). Compound loadings of 0.1, 0.5, 2.5, 5, 10, 25, and 50% content and an ethanolamine-blocked control bead (no compound) were investigated. A 50% bead loading gave the highest level of PK identification for both S1 and S2, extracting 34 and 55 PKs, respectively, from a single cell lysate. Control beads allowed overall identification of 23 PKs, which we term the kinase beadome, whereas sepharose-supported sunitinib (S7; 50% loading) identified 20, 11 of which were common to the control beads. The reliability of bead pull-downs was examined in duplicate experiments using two independently synthesized batches each of S1 and S2. Bead S1 showed high similarity in the absolute numbers of PKs identified across two experiments, at 40 and 35 PKs, of which 26 were common across the two batches of beads, with 14 and 9 unique PKs identified in each experiment. The S2 beads extracted 61 and 64 PKs with 55 PKs common across the two bead batches examined. We also report on the development and use of a novel promiscuous PKI analogue, 2-[(5-chloro-2{[4-(piperazin-1-yl)phenyl]amino}pyrimidin-4-yl)amino]-N-methylbenzene-sulfonamide (S15), which extracted 12 additional unique PKs over the two parent compounds from which it was designed, the combination of which identifies 160 unique PKs. S15 was based on the common pyrimidine core scaffold of S9 and S10. Thus, S15 expands the utility of kinobeads by broadening the kinome coverage for bead-based pull-down. Combining the data for all beads across 90 and 180 min liquid chromatography–mass spectrometry (LC–MS)/MS analysis identified a total of 160 unique PKs.