Drug use during pregnancy constitutes a major preventable worldwide public health issue. Birth defects, growth retardation and neurodevelopmental disorders are associated with tobacco, alcohol or drugs of abuse exposure during pregnancy. Besides these adverse health effects, drug use during pregnancy also raises legal and social concerns. Identification and quantification of drug markers in maternal and newborn biological samples offers objective evidence of exposure and complements maternal questionnaires. We reviewed the most recent analytical methods for quantifying drugs of abuse, tobacco, alcohol and psychotropic drugs in maternal, newborn and maternal-fetal unit biological samples by gas and liquid chromatography coupled to mass spectrometry. In addition, manuscripts comparing the usefulness of different biological samples to detect drug exposure during pregnancy were reviewed.