A specific and sensitive liquid chromatography-electrospray ionization mass spectrometry (LC–ESI–MS) method was developed for the determination of free and total ropivacaine in human plasma. The work-up procedure involved a simple precipitation of plasma proteins with methanol. Etidocaine served as the internal standard. After microscale equilibrium-dialysis, measurement of free ropivacaine levels was performed after direct injection of the dialysate into the chromatograph. The system used a Zorbax eclipse XD8 C8 analytical column packed with 5 μm diameter particles as the stationary phase. The mobile phase consisted of a 15-min gradient (mobile phase A: 0.05% (v/v) trimethylamine in acetonitrile, mobile phase B: 2 mM ammonium formate buffer (pH 3)). Mass spectrometric data were acquired in single ion monitoring mode at m/ z 275 for ropivacaine and m/ z 277 for etidocaine. The drug/internal standard peak area ratios (plasma) or peak areas (dialysate) were linked via a quadratic relationship to concentrations. Precision ranged from 1 to 7.6% and accuracy was between 92.6 and 109%. The lower limits of quantitation were 1 μg/l in plasma and 2 μg/l in the dialysate. This method was found suitable for the analysis of plasma samples collected during a clinical trial performed in 30 infants undergoing epidural anaesthesia or continuous psoas compartment block.