Methoxphenidine (MXP) is new psychoactive substance belonging to the group of dissociative anesthetics. It appeared on the black market in 2013, and MXP intoxications have been reported with at least three deaths since then. To understanding the mechanism of toxicity and effects of MXP, it is important to know its pharmacological properties (e.g. toxicity, activity on receptors and ADME) and metabolism which may be further exploited to study its mechanism of action in vivo. Metabolism of such compound may be useful not only in toxicological screenings, but together with pharmacokinetic and pharmacodynamic data it enhances the knowledge how these substances may affect the living organism. It is still very little information on the MXP and a method for analysis of biological samples (e.g. brain, liver, lungs and serum) remain to be developed. Standards of MXP, main metabolite norMXP and internal standard of deuterated MXP (MXP-d3) was prepared in-house and used as external calibrators. For determination of MXP and norMXP in biological samples, UHPLC-MS was used. Quantitative analyses were performed using LC system Dionex Ultimate 3000 (Thermo-Fisher, MA, USA) and the chromatographic separation was performed using Poroshell 120, Phenyl Hexyl (2.1 × 100 mm, 2.7 μm) column (Agilent, CA, USA). Mobile phases consisted of 0.1% formic acid and 10mM ammonium formate in 5% methanol in water (v/v) (A) and in 5% acetonitrile in methanol (v/v) (B) and were used at a flow rate of 0.4mL/min. The analytes were detected in multiple reaction monitoring (MRM) mode using QTRAP 6500+ (AB Sciex, MA, USA) with a Turbo V Ion source operating in positive electrospray (ESI + ) mode. Wistar rats given 20 mg/kg MXP were used as real samples. At 0; 0.5; 1; 2; 4; 8 and 24 hours after administration, samples of livers, lungs, brain and 2 mL of blood serum were collected from rats. There were 8 samples from each tissue at each time point. To extract analytes from these complex biological matrices, protein precipitation and salting-out liquid-liquid extraction (SALLE) were used. Due to the complex matrix, it was important to develop an extraction method with sufficient yield and low matrix effect. Liquid-liquid extraction, SALLE and protein precipitation were tested for each type of biological sample separately. These methods were selected on the basis of a previous study of structurally similar substances. Protein precipitation was chosen as the best extraction method for processing blood serum samples and SALLE was chosen for preparation of three other tissues. All validation parameters met the guidelines from the US Food and Drug Administration (FDA). 8 samples of 4 biological samples were analyzed in 7-time intervals. Based on these data, MXP and norMXP concentration curves were generated. A quantification and extraction method for the determination of MXP and the major metabolite norMXP in blood serum, brain, liver and lung samples from Wistar rats was developed and optimized. Pharmacokinetic profiles for MXP and norMXP in all 4 types of biological material used, were established. This work was a part of the research project supported by the Ministry of Health of the Czech Republic (grant No. NU21-04-00307) and also it was supported from the grants of Specific university research–grant No. A2_FCHI_2022_003 and A2_FPBT_2022_023.