Abstract High-throughput screening of large combinatorial chemical libraries in biochemical assays will benefit from reduced reagent volume and increased speed of measurement. Standard assays typically are performed in 96-well microtiter plates having 200-μL well volumes and up to an hour of incubation time. In this paper, we demonstrate a technique for precise and rapid measurement of the progress of an enzymatic reaction and its inhibition with reduced volume and time (for this work, the assay was mixed at the 200-μL level and detected in 2-μL volumes with minutes of total assay time). Directly measuring the enzyme activity in the small volume format yields a precise value for the median inhibitory concentration (IC 50) of an inhibitor compound. The model assay is the endoproteolytic cleavage of a small fluorogenic peptide by human neutrophil collagenase (MMP-8). The fluorogenic peptide was labeled at one end with a UV/blue fluorophore ( N-methylanthranilyl) and at the other end with a quencher (dinitrophenol). To generate inhibition data, a hydroxamate peptide analog inhibitor of collagenase, actinonin, was included in the reaction. The experiments were performed using ultraviolet laser illumination (325 nm wavelength) and parallel fluorescence detection by a cooled, charge-coupled-device camera system to increase sensitivity and speed. The assay volume was reduced to 2 μL for data collection, and the total time for mixing, incubation, and measurement was less than 6 min. For comparison to a standard format, the same assay was performed in a 96-well microtiter plate in 200 μL using 30 min of incubation and measurement in a microtiter plate fluorimeter. Median inhibitory concentrations (IC 50) for actinonin of 73 ± 16 and 100 ± 14 nM were obtained in the 2- and 200-μL assays, respectively. One concern with assay miniaturization and increases in throughput is a potential loss of precision and accuracy. Laser excitation and parallel detection of fluorescence is a promising approach for increased speed and reduced cost without loss of precision for proteinase inhibition assays.