To study the transfer of drugs from plasma into human milk [milk/plasma (M/P) ratio], we developed an in vitro method using paracetamol as the standard. The results obtained after equilibrium dialysis between milk and plasma were correlated with those obtained in vivo. For the in vivo study, 8 healthy postpartum women were enrolled as volunteers on the third postpartum day. After the ingestion of paracetamol 650mg, the in vivo comparison of milk and plasma area under the concentration-time curves (M/PAUC ratio) yielded a ratio of 0.94. In vitro determination 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 found in vivo. A strong correlation was found between plasma and milk concentrations obtained by both in vitro (r = 0.96, p < 0.0001) and in vivo procedures (r = 0.89, p < 0.001). These results demonstrate that the in vitro determination of M/P ratios for drugs that transfer in milk by passive diffusion, is similar to the data obtained in vivo. We conclude that this method can be used to avoid ethical constraints associated with the in vivo determination of human milk and plasma relative concentrations.