Choanoflagellates, the closest living relatives of animals, express diverse animal genes and may thereby provide insights into the ancestral roles of genes essential for modern animal cell biology and development. While efforts to study conserved gene families in choanoflagellates have been constrained by the relative inefficiency of currently available genetic tools, small molecule approaches are readily available and may provide a complementary and scalable approach for studying protein function. To study the physiological roles of choanoflagellate kinases, including animal kinase homologs, we established a high-throughput platform to screen the model choanoflagellate Salpingoeca rosetta with a curated library of human kinase inhibitors. We identified 36 diverse kinase inhibitors that disrupt S. rosetta cell proliferation. By exploring structure-activity relationships of one inhibitor, sorafenib, we identified a p38 kinase as a stress-activated regulator of cell proliferation in S. rosetta. This finding indicates a conserved p38 function between choanoflagellates, animals and fungi. Moreover, this study demonstrates that existing kinase inhibitors can serve as powerful tools to examine the ancestral roles of kinases that regulate modern animal development.