The purpose of our study was to develop original precolumn procedures and C18 high-performance liquid chromatography with a photodiode array detection method for the simultaneous determination of sixteen physiologically important cannabinoids and free cholesterol in plant and animal samples. The precolumn procedure for cannabinoid analysis in nonanimal samples involved two methanol and two n-hexane extractions due to the stronger extraction properties of the methanol solvent. The precolumn procedure in samples of animal origin (homogenized animal tissues, egg yolk of hens and feces), where cholesterol was analyzed together with cannabinoids, consisted of four n-hexane extractions due to the solubility of cannabinoids and cholesterol in n-hexane solvent. Methanol and n-hexane proved to be the appropriate solvents for the extraction of cannabinoids and cholesterol, which occur in assayed biological samples in wide concentration ranges. All the samples were chromatographically analyzed via gradient elution with acetonitrile with formic acid, water with formic acid and methanol. Our chromatographic method, irrespective of the precolumn procedure used, allowed for the selective, precise, accurate and sensitive quantification of cannabinoids and cholesterol in standard solutions and plant and animal samples. The sensitive and selective quantification of cannabinoids is important, especially in food products, because accurate monitoring of psychotropic cannabinoids is particularly necessary.