Kinases continue to be one of the most important targets in today's drug discovery efforts. Following the identification of lead compounds through screening efforts, it is important to profile these leads against other kinases within that family, as well as from other families, to ascertain potential off-target effects. Because many kinase assays require the use of different substrates, optimization time and costs during profiling can be prohibitive. Here we demonstrate the versatility of a luminescent ADP accumulation assay, where one set of reagents can be used for a wide variety of kinases with differing K(m app) for ATP and substrates. Assay sensitivity allows for the use of low enzyme concentrations and small percent ATP conversion levels while still maintaining high signal:background ratios. We have used a simple, inexpensive automated pipetting system to automate the entire process from enzyme optimization through generation of compound IC(50) values. Agreement with literature values proves this combination of chemistry and instrumentation provides a simple, yet robust solution for automated kinase profiling.