The main metabolites of methoxpropamine (MXPr) and the evaluation of their formation and excretion over time, using biological samples from mice, was investigated. MXPr is an arylcyclohexylamine dissociative drug structurally similar to 3-MeO-PCE, ketamine and deschloroketamine, which was recently identified on the European illegal market and added to the class of New Psychoactive Substances (NPS). Forensic laboratories are often required to identify new drugs and their metabolites, albeit the latter are yet not known or their reference standards are lacking. Therefore, we performed the investigation of methoxpropamine (MXPr) metabolism, in order to elucidate the distribution of the parent drug and its metabolites in urine and plasma over time. Furthermore, body hair was collected and analysed. Initially, urine samples from 16 mice were collected every hour for 6 consecutive hours. Furthermore, one sample was collected after 12 and 24 hours from the administration of increasing doses of MXPr (1-3-10 mg/kg). After 16 days of wash out, two doses of 1 and 3 mg/kg were administered and plasma samples were collected. After adding ketamine-d4, urine and plasma were diluted 1:3 with methanol/acetonitrile (95:5) and 5 μL were injected into the UHPLC-QTOF-HRMS. Based on the typical phase I and II metabolic reaction, the metabolic pattern was hypothesized. The tentative metabolites were identified by means of the fragmentation patterns, the exact masses of both their precursor and fragment ions. Finally, body hair collected one month before and after the injection of MXPr was analyzed following a specific procedure for keratin matrix. A wide array of metabolites was identified in urine, including normethoxpropamine, desmethylmethoxpropamine, dihydromethoxpropamine, desmethyl-normethoxpropamine, dihydro-desmethylmethoxpropamine and phase II glucuronide conjugates. Furthermore, the metabolic pathway that produces desmethylmethoxpropamine-glucoronide was identified as the preferential pathway for the elimination of MXPr from the body. In fact, 1 h after the administration, an abundant peak of excretion was observed with an average intensity 16 times higher than the free phase desmetyl-MXPr. The presence of normethoxpropamine (NorMXPr) in urine and plasma is also relevant, following the typical metabolic pathways based on dealkylation. Since norketamine is known to be pharmacologically active, so it may be for NorMXPr, thus probably contributing to the toxicity of this substance. In addition, desmethylmethoxpropamine-glucoronide, together with other minor products of metabolism, were detectable in urine up to 24 h after injection. Finally, MXPr and the major phase I metabolites were observed also in plasma (average concentration for the parent drug: 1.0 ng/mL) and hair (average concentration for the parent drug: 0.18 ng/mg). Few phases I and II metabolites were confirmed to be present in body hair, approximately one month after body administration. Some interesting differences were observed in the metabolites generated over time between female and male mice, a phenomenon that could be related to sexually dimorphic metabolism, both involving phase I and phase II enzymes. A comprehensive workflow for the detection of MXPr and its I and II phase metabolites are presented. Data acquisition included a TOF-MS high-resolution scan combined with TOF-MS/MS acquisition and demonstrated a considerable capability to detect and identify target compounds with high resolution levels. Thanks to this study, the identification of MXPr metabolites and their excretion times from the body was possible. The introduction of new target compounds will improve the efficiency of toxicological screening analysis on real samples and will extend the window of detection of the dissociative drug MXPr in biological matrices. In the future, human urine samples will be analyzed to confirm the detectability of the tentative metabolites.