Due to the growing public interest about the antitumor potential of tetrahydrocannabinol (THC) and its possible role as an agent for alleviating unpleasant symptoms of chemotherapy with irinotecan, some oncology patients reach for unregistered THC preparations. In order to evaluate the effect of THC on irinotecan metabolism in a rat experimental model, it is necessary to provide a sensitive, accurate and precise analytical method. This method was used for the determination of the mass concentration of THC and its metabolites [11-hydroxy-delta-9-tetrahydrocannabinol (THC-OH) and 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH)] in the rat urine of a group where only THC was applied and one where THC was applied simultaneously with irinotecan. For this purpose, optimized hydrolysis conditions and extraction of the investigated analytes were performed and the development and validation of the gas chromatographic method were done by detecting mass spectrometry (GC/MS) for the simultaneous determination of all three of the analysed analytes. When optimising the conditions of hydrolysis for THC, THC-OH and THC-COOH glucuronide, the best hydrolysis, so-called tandem hydrolysis, was demonstrated by β-glucuronidase enzyme from E. coli at 50 ° C for 2 hours followed by alkaline hydrolysis. When extraction was carried out, the solid phase extraction method was used for the cannabinoid columns that preceded the GC/MS analysis. The proposed method analysed the content of all three analytes in 24-hour rat urine (n = 10). THC was not detected in either sample, THC-OH was detected in 50% of samples, and THC-COOH in all of the samples. The method described in the paper is suitable for determining the mass concentration of THC and metabolic rate in the urine of the rat due to its susceptibility (detection limits: 0.8-1 μg/L), accuracy (> 96%) and precision (RSD < 6%).