This study investigates the transfer 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 ("yoked & forced") or voluntary ("freely-chosen") 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 a voluntary drinking group, an 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 <12%, with recovery of 86.2-118.8%. Results showed the presence of nicotine in 67% of samples from freely-chosen consumption group (1.30 ± 0.31 ng/mL) and in 60% of samples from yoked-consumption group (1.19 ± 0.62 ng/mL); cotinine was found in all the samples from freely-chosen (1.92 ± 0.77 ng/mL) and yoked-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.