Firefly luciferin methyl ester is hydrolyzed by monoacylglycerol lipase MAGL, amidase FAAH, poorly-characterized hydrolase ABHD11, and hydrolases known for S-depalmitoylation (LYPLA1/2), not just esterase CES1. This enables activity-based bioluminescent assays for serine hydrolases and suggests that the ‘esterase activity’ responsible for hydrolyzing ester prodrugs is more diverse than previously supposed.