A combination of high performance liquid chromatography (HPLC) and mass spectrometry (LC/MS) has proven its status as the most powerful analytical tool for screening and identifying drug metabolites in modern drug discovery. These techniques have become irreplaceable for drug metabolism laboratories, providing high amounts of information from a wide variety of samples. This review focuses on the most common and useful applications of these techniques when working on in vitro metabolism, more specifically with screening and identification of chemically stable or reactive metabolites formed via biotransformation reactions. Matching specific tasks and suitable instruments is a recurring consideration; for many reasons, the time-of-flight or orbitrap mass spectrometry provides clearly increased efficiency in metabolite profiling compared to other types of mass spectrometry.