This study investigates the passage of nicotine from lactating dams to their offspring through breast milk in the frame of a research focused to ascertain toxicological and neuro-behavioural effects on pups as consequence of either unavoidable (“passive”) or voluntary (“active”) maternal nicotine exposure. To this aim, plasmatic concentrations of nicotine and cotinine were determined by LC-MS/MS in Wistar rat pups whose mothers were orally administered with nicotine during lactation. Mothers were divided into voluntary consumption group, unavoidable consumption group, and controls. The limits of detection and quantification of the LC-MS/MS method were 0.20 and 0.65 ng/mL, respectively. Within-laboratory reproducibility (CV%) was <10.0%, with recovery of 86.2-118.8%. Results showed the presence of nicotine in 67% of samples from voluntary consumption group (1.30±0.31 ng/mL) and in 60% of samples from “passive” consumption group (1.19±0.62 ng/mL); cotinine was found in all the samples from voluntary (1.92±0.77 ng/mL) and “passive” consumption groups (1.43±0.30 ng/mL). Data provide an evidence-based support to maternal/offspring nicotine transfer as function of different ways of oral exposure.