Meconium, the first neonatal feces, is considered the "gold standard" for detecting prenatal drug exposure; however, several questions remain unanswered, including the efficacy of meconium testing compared to maternal self-report, drug detection windows, appropriate analytes of interest, maternal dose-meconium concentration relationships and prediction of adverse infant outcomes by meconium quantitative and/or qualitative results. Several analytical methodologies, offering significant improvements over existing procedures, were developed and validated to quantify drug and tobacco biomarkers in meconium. Toxicological results of meconium specimens obtained through national and international collaborations were compared to maternal self-report and biological testing results, and neonatal outcome parameters. Contrary to previous investigations of prenatal cocaine and opioid use, maternal self-report was more important than meconium testing for identifying prenatal cannabis and methamphetamine exposure. Meconium was previously assumed to reflect second and third trimester use, but objective evidence from maternal urinalysis throughout gestation suggest that the actual drug detection window spans only the third trimester. Novel biomarkers of tobacco, methamphetamine and cannabis were identified in meconium, improving the identification of affected infants. Furthermore, cleaving glucuronide conjugates was necessary to maximize drug-exposure detection for cannabinoids, but not tobacco biomarkers. Meconium concentrations observed in meconium were not related to maternal dose, but tobacco biomarker concentrations greater than 10 ng/g differentiated active smokers from passive or non-exposed women. Higher biomarker concentrations in meconium did not predict more severe neonatal outcome. Yet, the presence of a tobacco or cannabis biomarker in meconium was associated with fetal growth deficits. Also, opioid-positive meconium in methadone-exposed neonates indicated increased risk of prematurity and longer hospital stays. These data reinforce the need to evaluate the disposition of drug classes in meconium individually. Also, if maternal drug use in the first or second trimester is suspected, another means of objectively identifying exposure is necessary. Quantitative analyses in meconium may not be necessary, as no concentration effects were observed, but all assays employed for meconium testing should adhere to clinical and forensic standards of quality assurance.