Botulinum neurotoxin (BoNT) is a potent and specific biomolecule that is both implicated as a potential threat in bioterrorism and used in therapeutics. Highly sensitive and robust assays that measure BoNT activity are needed to manage outbreak or controlled distribution of BoNT. Current in vivo and in vitro assays have limitations, including high costs and variability for mouse bioassays, extensive preparations for primary and stem cell–derived neurons, and inherent low sensitivity for cell lines. Sensitivity of cell lines can be increased by direct differentiation and with their physiological relevance (compared with cell-free strategies) and robustness (compared with primary cell strategies); adopting cell lines is an attractive alternative to in vivo assays. Here, we present two distinct strategies that improved sensitivity of a cell line to BoNT serotype A (BoNT/A) without direct differentiation. We developed a cell-based BoNT assay using microscale culture and coculture of neuronal and Schwann cell lines, NG108-15 and S16, respectively, to improve both sensitivity and physiological relevance. Results showed that NG108-15 and S16 coculture decreased EC50 from 12.5 to 0.8ng/µl (p < 0.001) in macroscale and from 2.6 to 1.1ng/µl (p = 0.006) in microscale. In addition, NG108-15 monoculture at microscale decreased EC50 from 12.5 to 2.6ng/µl (p < 0.001) compared with macroscale. Finally, controlling the spatial arrangement of microscale coculture revealed that S16-derived soluble factors can increase sensitivity. Thus, our study demonstrates two distinct strategies for increasing the sensitivity of a cell line to BoNT using coculture and microscale culture, thereby advancing assay technology for BoNT detection.