New psychoactive substances (NPS) are modified, or synthesized drugs designed to mimic pharmacological effects produced by classical illicit substances. Fentanyl analogs are a group of opioid-type NPS that are ever-present in the public and have remained the cause of increasing overdoses and fatalities year after year.1 Whether simple or complex modification are performed on the core fentanyl structure, these NPS can become several fold more potent and produce lethal sedative effects.2 Ideally, published literature and testing standards are relied upon for detection and quantitation of illicit substances. However, the ambiguity involved with each emerging NPS becomes a serious threat for toxicologists as there can be little or no previously reported data or standards to aid in acquisition methods. In addition to this, the metabolite panels of some NPS are not entirely mapped out, which can result in metabolites that are unaccounted and undetected in analytical screenings. Since there are no standardized methods of acquisition for NPS and varying trends in prevailing NPS vary, there is a great need for extensive biotransformation studies. This study takes a multidimensional approach on the Phase I metabolism of two NPS fentanyl analogs, para-chloroisobutyryl fentanyl and despropionyl para-fluorofentanyl, via human liver microsomes (HLM) and electrochemical oxidation (ECO) assays and an in silico metabolism prediction tool, MetaSite. HLMs contain high concentrations of metabolizing enzymes, known as cytochrome P450 enzymes. ECO metabolism studies investigate redox reactions that may occur on the structure of an analyte through heterogenous electron transfers. These experiments involve a three-electrode setup. The Metasite metabolism prediction tool utilizes a unique algorithm that is independent of the training dataset to predict sites of metabolism and biotransformation based on chemical reactivity with CYP450 isoforms. Metabolism of pCIBF and DpFF was successfully predicted utilizing MetaSite’s liver enzyme model and 40 common CYP reaction mechanisms in addition to 5 uncommon reaction mechanisms. 25 total primary metabolites were produced for pCIBF. Among these were hydroxylated, dealkylated, and dehydrogenated metabolites. In silico metabolism of DpFF resulted in 24 predicted primary metabolites, of which were hydroxylated, dealkylated and carbonylated products. Each NPS was subjected to a second round of biotransformation to form 20 and 25 secondary metabolites for pCIBF and DpFF, respectively. Each metabolite’s structure, exact mass, and biotransformation type were documented for later comparative assessment. HLM experimental cofactors such as NADPH, glucose-6-phosphate, and magnesium chloride were added to a sodium bicarbonate buffer solution, at physiological pH, containing NPS and incubated for 4 h at physiological body temperature. ECO experiments involved the use of a gold, platinum, and glassy-carbon working electrode which delivers a potential, that is measured by a saturated calomel reference electrode, to a platinum/titanium counter electrode. Utilizing full-scan in LC-QToF-MS, a preliminary screening was completed, and the potential encountered metabolites were documented. Further analysis and confirmation were performed on the presumptive metabolites, via structural elucidation of MS/MS fragmentation for each metabolite. The in vitro assays conducted in this study allowed for the screening and confirmation of parent pCIBF and DpFF, as well as their metabolites. A total of 8 metabolites were produced for pCIBF and 11 metabolites for DpFF. Metabolites were categorized compared with results produced in silico. Several metabolites not previously reported were discovered in this study. These results suggest that ECO and in silico analyses of oxidative metabolites may be useful complements or alternatives to common HLM assays for comprehensive profiling of NPS metabolites in forensic toxicology.