The use of alcohol and tobacco is prevalent among pregnant women despite the well-known adverse effects of these substances imposed on the developing fetus and immense public health education efforts. Confirmation of gestational exposures to these compounds have relied mostly on maternal self-reporting, which is often underestimated because of guilt, embarrassment, and fear of punitive action. The presence of fatty acid ethyl esters in various biological matrices as a result of alcohol consumption initiated the development of neonatal screening tests for these emerging biological markers in meconium and hair. The levels of nicotine and cotinine in hair have long been used as objective indices for the quantification of exposure to active and passive smoking. Maternal segmental hair analysis in the study of pharmacokinetic changes in nicotine metabolism in the obstetric population is a novel application of this traditional analytical method. The latest developments and novel applications of meconium and hair testing for the confirmation of prenatal alcohol and tobacco exposure are discussed in this review.