Drug transport and metabolism are critical determinants of drug absorption, metabolism, distribution, elimination (ADME) as well as drug-drug interactions (DDIs) and toxicity. While significant progress has been made to utilize in vitro models to predict drug ADME and toxicity including physiologically-based pharmacokinetic and toxicokinetic modeling, these models are either not sufficiently complex or require comprehensive physiological data. ADME and toxicity biomarkers that can be quantified using accessible biofluid such as urine and blood from patients, healthy volunteers or preclinical species are recognized as a relatively non-invasive approach to facilitate DDI and toxicity potential of drugs. Recent data on biomarkers of drug transporter, metabolism, and toxicity in human and preclinical species suggest their promising applications in drug development.