Drug-drug interactions comprise a significant cause of morbidity and mortality worldwide as they may lead to adverse clinical events, result in decrease/inactivation of the therapeutic effect of a drug, may enhance drug toxicity and indirectly compromise treatment outcomes and adherence. Drug transporters and drug metabolism enzymes govern drug absorption, distribution, metabolism, and elimination (ADME). Inhibition or induction of transporter and drug metabolism enzymes can alter the ADME of a co-administered drug, which may lead to drug-induced toxicity or lack of efficacy. This review assesses our current understanding of the in vitro methods of evaluating CYPs and transporter-mediated DDI. The DDI prediction models based on in vitro assays are also discussed in this review. The applications, advantages and limitations of each method are also addressed in this review.