Over the last two decades, the role played by phytocannabinoids and endocannabinoids in medicine has gained increasing interest in the scientific community. Upon identification of the plant compound Δ9-tetrahydrocannabinol (THC) and of the endogenous substance anandamide (AEA), different methodological approaches and innovative techniques have been developed, in order to evaluate the content of these molecules in various human matrices. In this review, we discuss the analytical methods that are currently used for the identification of phytocannabinoids and endocannabinoids, and we summarize the benefits and limitations of these procedures. Moreover, we provide an overview of the main biological matrices that have been analyzed to date for qualitative detection and quantitative determination of these compounds. Copyright © 2013 John Wiley & Sons, Ltd.