Abstract A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and established for the quantitative determination of monocyte locomotion inhibitory factor, a pentapeptide (Met-Gln-Cys-Asn-Ser) produced by Entamoeba histolytica in axenic culture, in dog blood. The main challenge was the chemical and enzymatic instability of the peptide which was successfully overcome. After a simple protein precipitation, MLIF was separated from AS-5 (Met-Gln-Gly-Asn-Ser), acted as an internal standard, on a Gemini C18 column (5μm, 50mm×4.6mm i.d.) using a gradient elution of acetonitrile (0.2% formic acid) and water (0.2% formic acid) and detected by electrospray ionization tandem mass spectrometry. Excellent linearity was achieved (r>0.9943) over the linear range 5–1000ng/ml using 0.2ml blood sample. The validation results demonstrated that this method was specific, accurate and precise. It was successfully applied in measuring MLIF following intravenous infusion its administration at 0.2, 0.4, and 0.8mg/kg in beagle dogs to support the pre-clinical pharmacokinetic study.