Abstract Miniaturizing bioassays to the nanoliter scale for high-throughput screening reduces the consumption of reagents that are expensive or difficult to handle. Through the use of acoustic dispensing technology, nanodroplets containing 10 μM ATP (3 μCi/μL 32P) and reaction buffer in 10% glycerol were positionally dispensed to the surface of glass slides to form 40-nL compartments (100 droplets/slide) for Pim1 (proviral integration site 1) kinase reactions. The reactions were activated by dispensing 4 nL of various levels of a pyridocarbazolo-cyclopentadienyl ruthenium complex Pim1 inhibitor, followed by dispensing 4 nL of a Pim1 kinase and peptide substrate solution to achieve final concentrations of 150 nM enzyme and 10 μM substrate. The microarray was incubated at 30 °C (97% R h) for 1.5 h. The spots were then blotted to phosphocellulose membranes to capture phosphorylated substrate. With phosphor imaging to quantify the washed membranes, the assay showed that, for doses of inhibitor from 0.75 to 3 μM, Pim1 was increasingly inhibited. Signal-to-background ratios were as high as 165, and average coefficients of variation for the assay were ∼20%. Coefficients of variation for dispensing typical working buffers were under 5%. Thus, microarrays assembled by acoustic dispensing are promising as cost-effective tools that can be used in protein assay development.