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Botulinum neurotoxin (BoNT) causes the disease botulism, which can be lethal if untreated. Rapid determination of exposure to BoNT is an important public health goal. Previous work in our laboratory focused on the development of Endopep-MS, a mass spectrometry-based endopeptidase method for detecting and differentiating BoNT in buffer. This method can rapidly determine the presence of BoNT in a sample and differentiate the toxin type of BoNT present but does not yield additional information about the subtype. We now describe here the application of Endopep-MS to detect BoNT A in a spiked milk sample. This work also describes subtype identification achieved through mass spectrometric analysis of the protein toxin itself and does not require the presence of DNA from the toxin-producing bacteria. Tryptic digests of A1 and A2 subtypes of BoNT were analyzed by mass spectrometry, and peptides unique to either the A1 or A2 subtype were subjected to tandem mass spectrometry analysis to confirm their identities. Finally, subtype identification through mass spectrometric analysis was performed on BoNT A isolated from spiked milk. In its entirety, this method would allow for analysis of BoNT with toxin type identification in a few hours and subtype identification within 24 h.