In this review, some well-established assays and more recent markers developed for the understanding of the biological activity of pharmaceutical drugs belonging to different pharmacological classes (nonsteroidal anti-inflammatory drugs, cardiovascular drugs, and central-nervous-system-acting drugs) are considered. The results of in vitro studies are reviewed and critically compared with those available from clinical trials, and their relevance for the elucidation of the mechanism of action of the drugs is discussed. Although from this examination a positive correlation between the in vitro and in vivo data seems to emerge, the small number of clinical trials available, their low number of patients enrolled, and sometimes the arbitrary or inappropriate choice of the biomarker(s) used highlight the need for (1) more standardized protocols to allow a reliable comparison of the results from different studies and (2) the development of new and more appropriate and specific biomarkers for the evaluation of oxidative stress before and after drug intervention.