The aim of the study was to develop a method of gas chromatography coupled to mass spectrometry (GC-MS) and liquid chromatography with photodiode array detection (HPLC-DAD) for the determination of non-derivatized neutral cannabinoids (CBDs-N), cannabinoids in the form of acids (CBDs-A) and cholesterol (Chol). Emphasis was also placed on the comparison of the GC-MS method with the HPLC-DAD method. A method was developed for the simultaneous determination of 16 physiologically significant cannabinoids (CBDs) by HPLC-DAD and 17 CBDs by GC-MS and free Chol by both methods in plant and animal samples. The pre-column procedure for the analysis of CBDs in plant samples included two extractions with methanol and two extractions with n-hexane. The pre-column procedure in samples of animal origin (homogenized animal tissues, hen egg yolk and feces), where Chol was analyzed together with CBDs, consisted of four n-hexane extractions. Methanol and n-hexane proved to be suitable solvents for the extraction of CBDs and Chol occurring in the tested biological samples in a wide concentration range. All samples were analyzed by HPLC-DAD with gradient elution with acetonitrile with formic acid, water with formic acid and methanol. This method allowed for selective, precise, accurate and sensitive quantification of CBDs and Chol in standard samples and in plant and animal samples. Sensitive and selective quantification of CBDs is important, especially in food products, since the accurate monitoring of psychotropic CBDs is particularly important. A combination of a long temperature program and a gradual temperature increase was used in mass spectrometry to avoid problems resulting from peak overlap between CBDs and Chol. CBDs-A underwent decarboxylation to CBD-N during GC-MS analysis and thus, by direct injection, the response was a sum peak for both CBDs-N and CBDs-A forms. Extraction with NaOH removed the CBDs-A forms and only the neutral forms of CBDs could be analyzed. No losses of CBDs-N by NaOH solution were observed in the extracted samples. GC-MS analyses of samples before and after NaOH extraction allowed for the quantification of CBDs-A and CBDs-N. The use of the C18 HPLC-DAD method allowed for the simultaneous determination of CBDs-N and CBDs-A. Compared to the C18 HPLC-DAD method, the GC-MS technique offered improved sensitivity, accuracy, specificity and sufficient separation of non-derivatized CBDs and Chol from biological materials of endogenous origin, especially in hemp and hen egg yolk samples. The scientific novelty of this study is the application of the GC-MS method for the quantification of non-derivatized forms of CBDs-A, CBDs-N and Chol.