A rapid, sensitive, and accurate analytical method was developed for determination of lactic acid (LA) in human plasma to monitor lactic acidosis. This method was based on an ultrasound-assisted emulsification microextraction (USAEME) method followed by gas chromatography with flame ionization detection (GC–FID). Derivatization of LA was carried out by a low density alcoholic solvent which performs both as an extraction solvent and derivatization agent, simultaneously. In this procedure, 100 μL of binary mixtures of pentan-1-ol with toluene (70 : 30, v/v %) was slowly injected into a 10 mL acidified aqueous sample of LA placed into an ultrasonic water bath. The resulting emulsion was centrifuged and after derivatization, 2 μL of organic phase was analysed by GC–FID. The effective variables were evaluated to optimize the efficiency of USAEME. Under the optimum conditions, good linearity in the range of 0.06–7.77 mmol L–1 was obtained with a correlation coefficient (R2) of 0.991 and a limit of detection (LOD) of 0.04 mmol L–1 for water samples. The inter-day and intra-day repeatability of the proposed method in human plasma were evaluated in terms of the relative standard deviation (RSD %) and were found to be <10 %. The results revealed that the USAEME–GC–FID method can be applied successfully for determination of LA in human plasma samples with satisfactory accuracy and precision.