Cyanide is a metabolic poison that inhibits utilization of oxygen to form ATP. The consequences of acute cyanide exposure are severe: toxic doses result in loss of consciousness, cardio and respiratory failure, hypoxic brain injury, and dose-dependent death from within minutes to hours. In a mass casualty scenario such as an industrial accident or terrorist attack, currently available cyanide antidotes would leave many victims untreated in the short time available for successful medical countermeasure administration. Therefore, there is a need for rapidly acting antidotes that can be quickly administered to large numbers of people who may be unconscious. Sulfanegen, a novel preclinical cyanide antidote, is being developed to meet this need. Sulfanegen is administered by intramuscular injection (IM), which has the advantages of requiring minimal training for first responders, as well as the potential for rapid antidotal administration to many affected people. Due to ethical issues involved with testing cyanide antidotes in human subjects, sulfanegen cannot go through the normal clinical trial path to drug approval. However, the FDA has published guidelines for evidence needed to gain approval under the “Animal Rule”, which this thesis attempts to address. The “Animal Rule” allows for new drug approval if the following conditions are met: 1) Effective in more than one animal model with a response predictive for humans, 2) Pharmacokinetics understood well enough to determine effective dose, and 3) A well-understood mechanism of action and pathology of the disease. v We first elucidated the animal models predictive for human efficacy testing by MST species comparison of the blood of many common laboratory animals. Based on this preliminary screen, species closest to that of humans were chosen to characterize the MST activities in metabolically active tissues. Using those findings, we determined swine were not an appropriate model for predicting efficacy in humans, while murine and rabbit models were appropriate. Next, we developed an isocratic UV-VIS HPLC method for characterizing the pharmacokinetics of sulfanegen in humans. Pre-column derivitization with NEM prevented the reformation of the 3-mercaptopyruvate (3MP) dimer and allowed for chromatographic detection. Moreover, this method is easily modified for applications to quality control, being able to separate sulfanegen from its precursor molecules and predicted decomposition products. Finally, we examined the potential secondary mechanisms of sulfanegen administration. MST catalyzed detoxification of cyanide to thiocyanate is the primary mechanism of action, but secondary mechanisms involving 3MP alone or in conjunction with MST contribute to the overall efficacy of sulfanegen. 3MP was found to be a potent antioxidant, capable of quenching cyanide induced ROS. Additionally, H2S generation by MST was confirmed after sulfanegen administration. Although it was confirmed that cyanohydrin formation was occurring, this probably does not contribute to reversing cyanide-induced cytochrome c oxidase inhibition.