A validated method for the determination of sufentanil in human plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is described. Sufentanil was extracted from human plasma with solid-phase-extraction using deuterated sufentanil, [(2)H(5)]-sufentanil, as internal standard. Sufentanil and the internal standard were determined with an API 4000 tandem mass spectrometer equipped with a Turbo-V-Source operating in positive ESI mode on an Alltima HP HILIC straight phase column. The method showed a lower limit of quantification of 0.25 pg/ml (12.5 fg on column). The applicability of the method is shown in a clinical study, in which levels of sufentanil in plasma of parturients and arterial umbilical plasma of their neonates following patient-controlled epidural analgesia (PCEA) under several regimen treatments was analyzed.