Accurate detection of botulinum neurotoxin (BoNT) requires assessing both its proteolytic SNARE-cleaving activity and its ability to inhibit synaptic transmission. While the mouse bioassay remains the traditional diagnostic standard, its prolonged turnaround time, limited reproducibility, and ethical constraints underscore the urgent need for advanced in vitro methodologies. Recent advances in analytical chemistry, stem-cell-derived neuronal platforms, and multi-omics profiling enable the development of sensitive systems that replicate BoNT’s molecular and functional mechanisms with greater precision than conventional in vivo models.

Aim. To evaluate current methodologies for the diagnosis and functional analysis of botulinum neurotoxin and, synthesizing these findings, to propose a conceptual algorithmic model for comprehensive neurotoxin detection independent of in vivo methods.

Materials and methods. A literature review was conducted utilizing the PubMed, Scopus, and Web of Science databases. Studies detailing molecular, cellular, multi-omics, and computational approaches to BoNT detection were included. The extracted data were synthesized across molecular, cellular, and systems-level domains to construct an integrated diagnostic model.

Results. Modern diagnostic strategies increasingly surpass the mouse bioassay in terms of sensitivity, specificity, and mechanistic insight. SNARE-specific proteolytic profiling, notably Endopep-MS, enables precise detection of BoNT serotypes and their functional states by quantifying cleavage kinetics across a diverse array of substrates. Complementary human iPSC-derived neuronal platforms allow for direct functional assessment, revealing electrophysiological suppression, altered calcium signaling, and reporter-confirmed SNARE cleavage within living cells. Multi-omics analyses, including single-cell transcriptomics, epigenomics, and metabolomics, capture early stress signatures, serotype-specific responses, and determinants of neuronal susceptibility. Additional resolution is provided by receptor-binding kinetics and intracellular trafficking studies, which elucidate how serotype-dependent variations dictate overall toxicity. The integration of these molecular, cellular, and systems-level insights establishes the foundation for the ULTIMA-BoNT framework, a unified platform designed for high-precision BoNT detection.

Conclusions. The convergence of proteolytic assays, physiologically relevant neuronal models, and multi-omics analytics presents a robust, reproducible, and ethically sustainable alternative to the mouse bioassay. This integrative approach not only provides profound mechanistic insights but also supports the predictive analysis of emerging BoNT variants.