Mesocarb metabolism in humans is the target of this investigation. A high-performance liquid chromatographic (LC) method with electrospray ionization (ESI)-ion trap mass spectrometric (MS) detection ion trap “SL” for the simultaneous determination of mesocarb and its metabolites in plasma and urine is developed and validated. Ten metabolites and the parent drug are detected in human urine, and only four in human plasma, after the administration of a single oral dose of 10 mg of mesocarb (Sydnocarb, two 5-mg tablets). Seven of this metabolites have been found for the first time. The confirmation of the results and identification of all the metabolites except amphetamine is performed by LC-MS, LC-MS-MS, and LC-MS3. In the case of doping analysis, the reliable detection time for mesocarb (long-life dihydroxymesocarb metabolites of mesocarb) is approximately 10–11 days after the administration of the drug, which is a significant increase over the existing data. The detection of amphetamine in plasma and urine is made using simple flow-injection analysis without a chromatographic separation. The addition-calibration method is used with plasma and urine. The mean recoveries from plasma are 49.2% and 57.4% for mesocarb concentrations of 33.0 and 66.0 ng/mL, respectively, whereas the recoveries from human urine are 76.9% and 81.4% for concentrations of 1 and 2 ng/mL, respectively. Calibration curves (using an internal standard method) are linear (r2 > 0.9969) for concentrations 0.6 to 67 ng/mL and from 0.05 to 5 ng/mL in plasma and urine, respectively. Both intra- and interassay precision of plasma control samples at 3, 40, and 55 ng/mL are lower than 6.2%, and the concentrations do not deviate for more than −3.4% to 7.3% from their nominal values. In urine, intra- and interassay precision of control samples at 0.08, 1.5, and 3.0 ng/mL is lower than 14.1%, with concentrations not deviating for more than −11.3% to 13.7% from their nominal values. The plasma disappearance curve of the parent drug is obtained. The major pharmacokinetic parameters are calculated.