A simple and reproducible method for the qualitative and quantitative analysis of free mono- and disaccharides (fructose, glucose, galactose, sucrose, lactulose and lactose) in milk-based formulae by high-performance liquid chromatography (HPLC) with refractive index (RI) detection was developed and validated. The method showed good linearity with determination coefficients exceeding 0.99. The limits of detection (DL) in these sugars were 0.17, 0.13, 0.06, 0.16, 0.05 and 0.25 mg/ml, respectively; and the limits of quantification (QL), 0.27, 0.24, 0.20, 0.26, 0.22 and 0.38 mg/ml. The relative standard deviations (R.S.D.s) for repeatability in fructose, sucrose, lactulose and lactose were 0.78, 0.99, 2.91 and 0.46 and the R.S.D.s for reproducibility were 4.8, 6.15, 7.04 and 2.49, respectively. Recoveries in all sugars were between 93 and 113%.