Cytochromes P450 (CYPs) play a central role in the Phase I metabolism of drugs and other xenobiotics. It is estimated that CYPs can metabolize up to two-thirds of drugs present in humans. Over the past two decades, there have been numerous advances in in vitro methodologies to characterize drug metabolism and interaction involving CYPs. Areas covered: This review focuses on the use of in vitro methodologies to examine CYPs role in drug metabolism and interaction. There is an emphasis on their current development, applicability, advantages and limitations as well as the use of in silico approaches in complementing and supporting in vitro data. The article also highlights the challenges in extrapolating in vitro data to in vivo situations. Expert opinion: Advances in in vitro methodologies have been made such that data can be used for in vivo prediction with comfortable degree of confidence. Improved assay designs and analytical techniques have permitted development of miniaturized assay format and automated system with improved sensitivity and throughput capacity. High-quality experimental designs and scientifically rigorous assessment/validation protocols remain crucial in developing reliable and robust in vitro models. With continued progress made in the field, in vitro methodologies will continually be employed in evaluating CYP activities in pharmaceutical industries and laboratories.