A rapid and sensitive homogeneous assay method has been developed for the determination of subtilisin. The method employs a protein substrate labelled with two fluorescent dyes with fluorescence energy transfer (FET) characteristics. The doubly-labelled substrate was prepared by chemically coupling bovine serum albumin with lucifer yellow and rhodamine dyes. The fluorescence emission from the lucifer labels was initially quenched due to the FET to the adjacent rhodamine labels. However, upon the addition of subtilisin into the labelled substrate solution, increased fluorescence was observed as the enzyme hydrolyzed the substrate and reduced the FET effect. The rate of increase in fluorescence due to substrate hydrolysis was used to calibrate the subtilisin assay. It was linear over the range 0–150 ng of the enzyme (n=8, r2=0.985). The assay was fast with a time of 30 sec to exceed the limit of detection (LOD) signal for 60 ng of subtilisin in 600 μl. In this volume, the LOD for the enzyme was 4.2 ng (99% confidence).