Pregnant women are exposed to benzodiazepines for therapeutic purposes during gestation. The goal of this study was to evaluate prenatal exposure to benzodiazepines. Time of exposure during course of pregnancy is a significant aspect of fetal exposure to drugs. Benzodiazepine concentration assay in hair of mothers and newborns exposed prenatally to these drugs was performed in the studies. Development, validation and evaluation of benzodiazepine determination method in mothers and their newborns enables assessment of health risks for the child and implementation of adequate therapeutic procedures. We used A LC-ESI-MS/MS method that allowed determination of diazepam (the main benzodiazepine used by pregnant women was diazepam) and its metabolites (nordazepam, oxazepam) in hair of mothers and newborns. LOQ 10 pg/mg of hair was used in the study. Results: concentration of nordazepam was higher than parent drug (diazepam) and higher in newborns’ hair when compared to mothers’. The mean concentrations of diazepam in mothers’ hair were 31.6±36.0 and 34.1±42.4 pg/mg in the second and third trimester of pregnancy respectively. The mean concentration of diazepam in newborns’ hair was higher and reached levels of 53.3±36.5 pg/mg. The mean concentration of nordazepam in the mothers’ hair corresponding to the second and third trimester was 52.9±48.1 and 89.9±122.8 pg/mg, respectively. Nordazepam in the newborns’ hair was detected at the mean level of 108.1±144.2 pg/mg. It was concluded that diazepam and nordazepam are permanently incorporated into the hair structure. Presence of diazepam and its metabolites in newborn’s hair confirms that these benzodiazepines permeate placental barrier. Segmental analysis of mothers’ hair enabled the assessment of drug administration time. Diazepam and its metabolites determined in hair of newborns may serve as biomarkers of prenatal exposure to these drugs. The performed LC-MS/MS analysis was accurate enough to determine even low concentrations of benzodiazepines, at the level of few pg/mg of hair. Levels of diazepam detected in hair of newborns were higher than levels determined in mothers. This may confirm the fact, that fetus’s ability to metabolize diazepam is scarce. Nordazepam was found in higher concentrations in hair of newborns than in hair of mothers, which may suggest that it is cumulated in child’s organism. Other metabolites of diazepam - oxazepam and temazepam - were detected in very few cases, in low concentrations.