Biological markers or biomarkers are measurable biological phenomena, which can be quantified and validated. Biomarkers are essential to the clinical practice of medicine, epidemiology, and biological research. Clinically, biomarkers are used for screening, diagnosis, monitoring treatment, and to support clinical decision-making. In research contexts, biomarkers facilitate the measurement of outcomes, serving as endpoints in clinical trials. In a substance use context, biomarkers are often used to directly or indirectly measure the consumption of a psychoactive substance. This chapter defines biomarkers and relevant statistical concepts and then goes on to discusses the most notable biomarkers for alcohol, nicotine, cannabinoids, opioids, and stimulants. The chapter closes with a review of the future directions of biomarkers, including genetic, epigenetic, neuroimaging, and neuroinflammation markers that are currently under research.