Cell-based assays are critically important for measurements of botulinum neurotoxin serotype A (BoNT/A) activity in field and biological samples, in-process samples, and formulated products, to reduce the need for in vivo testing of toxin activity, and to identify inhibitors of BoNT/A action. A well-designed cell-based assay should be able to evaluate, indirectly or directly, all three steps in BoNT activity: receptor binding, internalization and translocation, and catalytic activity. A new 96-well plate cell-based assay has been developed using differentiated Neuro-2a cells and a Western Blot (WB) read-out with a custom antibody to SNAP25197 that specifically recognizes the cleaved product of BoNT/A. The assay is based on a 7 point dose-response curve fitted with a 4 parameter logistic model that generated EC50 values of 2.9±0.36nM. The assay is run by five operators with Z′ values of 0.7 and signal-to-noise values greater than 100 fold. Further optimization of differentiation, BoNT/A treatment, and WB conditions resulted in increased sensitivity and EC50 values of 50–100pM. In conclusion, a new specific and robust medium-throughput screening Western Blot cell-based assay for measuring BoNT/A activity has been developed and characterized. Optimization of the assay resulted in increased sensitivity that at the moment is still insufficient for developing a potency assay for product release, but may be useful for evaluation of more concentrated samples.