Cannabis is the most widely used illicit drug in the world. The pharmacological properties of Δ(9)-tetrahydrocannabinol also make it a promising molecule in the treatment of different pathologies. Understanding the PKs and PDs of this drug requires the determination of the concentration of Δ(9)-tetrahydrocannabinol and metabolites in biological matrices. For this purpose many analytical methodologies using mass spectrometric detection have been developed. In recent years, LC-MS/MS has become the gold standard in analysis of tetrahydrocannabinol and its metabolites due to the high selectivity and sensitivity, but above all, due to the ability to determine free and conjugate analytes in one run.