Kinases represent an important class of targets for pharmaceutical drug development. Microfluidic devices capable of running kinase assays with either an on-chip or an off-chip enzymatic reaction have been developed. For the on-chip assay, reagent addition, mixing, enzymatic reaction, and electrophoretic separation and detection of substrate and product all take place in the channels of the microfluidic chip. For the off-chip assay, the reaction takes place in a microtiter plate, whereas the electrophoretic separation and detection of substrate and product take place in the channels of the chip. To probe differences between the on-chip and off-chip assays, a panel of commercially available kinase inhibitors was assayed at 10 microM against cyclic AMP-dependent protein kinase A, glycogen synthase kinase 3beta, mitogen- and stress-activated protein kinase, and Akt1 using both the off-chip and on-chip assays. Good correlation was observed between inhibition measured by the two methods, with most of the differences in measured inhibition being attributed to compound solubility and enzyme concentration effects. Microfluidic devices represent an attractive platform for kinase assays due to high data quality and the possibility of on-chip assay integration, leading to reagent and labor savings.