The “entourage effect” has drawn much attention to the individual and synergistic effects of major and minor cannabinoids in recent years. However, due to a lack of harmonized sample preparation and instrumental methods, it has been challenging to conduct well-controlled clinical studies and to compare results across labs. Biosynthesized cannabinoids produced from microorganisms, such as bacteria, yeast, and algae, have further challenged this task with their complex aqueous matrices. The objective of this study was to develop a purification procedure for such complex matrices and a quantification method for major and minor cannabinoids. Isochrysis galbana was used as the model matrix. After methanol extraction, samples were purified using solid phase extraction, derivatized with N-methyl-N-(trimethylsilyl)trifluoroacetamide, and analyzed using gas chromatography-mass spectrometry (GC-MS) in electron ionization mode. A strong anion-exchange column efficiently recovered the cannabinoids in acid form (olivetolic acid, cannabidiolic acid, tetrahydrocannabinolic acid, and cannabigerolic acid). A graphitized carbon black column was necessary to purify the neutral cannabinoids (olivetol, cannabidiol, and tetrahydrocannabinol). Both columns removed amino acids, sugars, fatty acids, alcohols, and pigments from the algae extract and prepared samples suitable for derivatization and GC-MS analysis. A sensitive and robust GC-MS method was developed to simultaneously quantify these acid-form and neutral-form cannabinoids. The method showed good linearity and reproducibility in the tested range (0.05 to 0.5 ppm in extract). This method has the potential to be a harmonized profiling tool for cannabinoids in complex matrices and to help with comparison among clinical studies.