A fluorescence-based continuous-flow enzyme affinity detection (EAD) setup was used to screen cytochrome P450 BM3 mutants on-line for diversity. The flow-injection screening assay is based on the BM3-mediated O-dealkylation of alkoxyresorufins forming the highly fluorescent product resorufin, and can be used in different configurations, namely injection of ligands, enzymes and substrates. Screening conditions were optimized and the activity of a library of 32 BM3 mutants towards the recently synthesized new probe substrate allyloxyresorufin was measured in flow-injection analysis (FIA) mode and it was shown that large activity differences between the mutants existed. Next, six BM3 mutants containing mutations at different positions in the active site were selected for which on-line enzyme kinetics were determined. Subsequently, for these six BM3 mutants affinity towards a set of 30 xenobiotics was determined in FIA EAD mode. It was demonstrated that significant differences existed for the affinity profiles of the mutants tested and that these differences correlated to alterations in the BM3 mutant-generated metabolic profiles of the drug buspirone. In conclusion, the developed FIA EAD approach is suitable to screen for diversity within BM3 mutants and this alternative screening technology offers new perspectives for rapid and sensitive screening of compound libraries towards BM3 mutants. © 2011 Society for Laboratory Automation and Screening.