Botulism, the disease caused by botulinum neurotoxins (BoNTs), secreted by the spore-forming, anaerobic bacteria Clostridium botulinum, has been associated with food poisoning for centuries. In addition, the potency of BoNTs coupled with the current political climate has produced a threat of intentional, malicious poisoning by these toxins. The ability to detect and measure BoNTs in complex matrixes is among the highest research priorities. However, the extreme potency of these toxins necessitates that assays be capable of detecting miniscule quantities of these proteins. Thus, signal-boosting strategies must be employed. A popular approach uses the proteolytic activity of the BoNT light chain (LC) to catalyze the cleavage of synthetic substrates; reaction products are then analyzed by the analytical platform of choice. However, BoNT LCs are poor catalysts. In this study, the authors used the osmolyte trimethylamine N-oxide (TMAO) to increase the proteolytic activities of BoNT LCs. Their data suggest that concentrated solutions of TMAO induce complete folding of the LCs, resulting in increased substrate affinity and enhanced enzyme turnover. The authors observed increases in catalysis for BoNT serotypes A, B, and E, and this increased proteolytic activity translated into substantial increases in analytical assay sensitivity for these medically relevant toxins.