© 2015 Future Science Ltd. Perinatal exposure to one or more drugs of abuse can affect the neonate temporarily or permanently. In addition to meconium, the evaluation of perinatal exposure to drugs of abuse has been achieved by testing biological matrices coming from the newborn (neonatal hair) and from the pregnant or nursing mother (maternal hair and breast milk). These matrices have the advantage of noninvasive collection and account for a sizable time window of active and passive exposure. Sensitive and specific analytical methods are required to determine minute amounts of drugs of abuse and metabolites in these matrices. The present manuscript reviews the newest analytical methods developed to detect drugs of abuse as well as ethanol biomarkers in maternal and neonatal hair and breast milk.