In present time the use or misuse of morphine and its derivatives are monitored by assaying the presence of the drug and its metabolites in biofluids. In the present review, focus is placed on the sample preparation and on the separation techniques used in the current best practices of bioanalysis of morphine and its major metabolites. However, as methods for testing the misuse of heroin, a morphine derivative, often involve bioanalytical methods that cover a number of other illicit drug substances, such methods are also included in the review. Furthermore, the review also includes bioanalysis in a broader perspective as analysis of plant materials, cell cultures and environmental samples. The review is not intended to cover all publications that include bioanalysis of morphine but is more to be considered a view into the current best practices of bioanalysis of morphine, its metabolites and other related substances.