Botulinum neurotoxins contain proteases that cleave specific intra-neural proteins essential for neurotransmitter release. Toxin types A, E and C1 intra-cellularly cleave SNAP25 resulting in a flaccid paralysis. As a consequence, various different endopeptidase assays have been developed to specifically detect the toxins enzymatic activity, however, many of these suffer from variability, low sensitivity or unwanted interference exerted by product specific excipients. The current studies utilised solid phase synthesized SNAP25(137-206) peptide substrate, and specific antibody to either the SNAP25(190-197) or (173-180) octapeptide epitopes that become exposed following cleavage by toxin types A or E respectively. Assay sensitivity was increased 50 fold by the use of an optimal 0.5% Tween 20 concentration in tandem to 0.1% albumin together with an improved, simplified assay design without a pre-activation / reduction step. Sensitivities capable of detecting 0.01 LD50/ml (40fg/ml or 0.3fM) of type A toxin was achieved with a linear dose response between 0.1 and 1 LD50/ml. This provides sufficient sensitivity and precision (inter assay GCV of < 2%) for monitoring activity within any current or newly marketed therapeutic products containing less units per vial and may also make it applicable for other applications. Both purified haemagglutinin free and complexed toxins could be detected equally. Unlike type A, type E activity could unexpectedly be detected in the complete absence of reducing conditions and the optimal assay had a limit of detection of 0.2LD50/ml (4.8pg/ml) with a linear dose response between 1 and 10LD50/ml. The principle of using a detecting antibody to a substrate sequence buried within the native substrates alpha-helix may be further expanded to other specific enzyme cleavage reactions in the future.