To study the transfer of drugs from plasma into human milk [milk/plasma (M/P) ratio], we developed anin vitromethod using paracetamol as the standard. The results obtained after equilibrium dialysis between milk and plasma were correlated with those obtainedin vivo.For thein vivostudy, 8 healthy postpartum women were enrolled as volunteers on the third postpartum day. After the ingestion of paracetamol 650mg, thein vivocomparison of milk and plasma area under the concentration-time curves (M/PAUCratio) yielded a ratio of 0.94.In vitrodetermination of the M/P ratios was carried out on plasma and milk taken on the third and 26th days after delivery. The M/P ratio obtained after equilibrium dialysis (0.91 ± 0.01) was in good agreement with that foundin vivo.A strong correlation was found between plasma and milk concentrations obtained by bothin vitro(r = 0.96, p < 0.0001) andin vivoprocedures (r = 0.89, p < 0.001). These results demonstrate that thein vitrodetermination of M/P ratios for drugs that transfer in milk by passive diffusion, is similar to the data obtainedin vivo.We conclude that this method can be used to avoid ethical constraints associated with thein vivodetermination of human milk and plasma relative concentrations.