The research of drug metabolism is essential for successful drug discovery. Phytochemicals, particularly those from herbal medicines, have the potential to affect diseases such as cancer, heart disease, diabetes, and high blood pressure, etc., showing great advantages in disease prevention and personalized treatment. In the past decade, phytochemical metabolism played a major role in the discovery of valuable leading compounds of new drugs. However, the metabolic studies of phytochemical compounds are extremely challenging due to numerous analogs with similar structures, complex endogenous matrices with multi-components in biofluids and cell or tissue extracts. For the feasibility of monitoring phytochemical metabolism, suitable bioanalytical tools and strategies have to be developed and validated. Here we critically review the literature from 2009 to 2014, and present generalized strategy for phytochemical metabolism in biological matrices, including the methodology of metabolism, sample preparation methods and detection techniques. In addition, the present paper covers the diverse aspects of metabolism studies on phenolic acids, flavonoids, anthraquinones, alkaloids, terpenoids, coumarins, lignans and steroids in medicinal plants. Finally, prospects for further research are also considered.