A sensitive and simple spectrophotometric method for quantification of lactulose without interference from aldoses was developed. The method was based on hydrolysis of lactulose under acidic conditions. The hydrolysed product reacts with cysteine hydrochloride-tryptophon reagent, giving an absorption peak at 518 nm. By following the conditions optimised in this paper, the absorbance value generated by lactose or galactose was far less than that of lactulose of the same amount, suggesting that the interference from aldoses for determination of lactulose could be neglected. The calibration curve was linear in the range of 5–25 μg mL−1 with a correlation coefficient of 0.999. The limit of detection of lactulose at 518 nm was 0.58 μg mL−1. The variation between the results for lactulose (18 μg mL−1) was 1.02%. These facts revealed that the method can be recommended for the quantitative determination of lactulose in case of syrups, biological fluids or dairy products.