The deleterious effects exerted by prenatal ethanol exposure include physical, mental, behavioural and/or learning disabilities that are included in the term fetal alcohol spectrum disorder (FASD). Objective assessment of exposure to ethanol at both prenatal and postnatal stages is essential for early prevention and intervention. Since pregnant women tend to underreport alcohol drinking by questionnaires, a number of biological markers have been proposed and evaluated for their capability to highlight gestational drinking behaviour. These biomarkers include classical biomarkers (albeit indirect) of alcohol-induced pathology (mean corpuscular volume (MCV), gamma glutamyltransferase (GGT), aspartate aminotransferase (AST) and alanine aminotransferase (ALT)) acetaldehyde-derived conjugates, and finally derivatives of non-oxidative ethanol metabolism (fatty acid ethyl esters (FAEEs), ethyl glucuronide (EtG), ethyl sulphate (EtS) and phosphaditylethanol (PEth)). Since ethanol itself and acetaldehyde are only measured few hours after ethanol intake in conventional matrices such as blood, urine and sweat, they are only useful to detect recent ethanol exposure. In the past few years, the non-oxidative ethanol metabolites have received increasing attention because of their specificity and in some case wide time-window of detection in non-conventional matrices from the pregnant mother (oral fluid and hair) and fetus-newborn (neonatal hair, meconium, placenta and umbilical cord). This article reviews bioanalytical procedures for the determination of these markers of ethanol consumption during pregnancy and related prenatal exposure. In addition, clinical toxicological applications of these procedures are presented and discussed.