Abstract: OBJECTIVE To establish UPLC-QDa for the content determination of lactulose and its related substances including tagatoes, fructose, galactose, epilactose, lactose in lactulose oral solution. METHODS ACQUITY UPLC BEH Amide column(2.1 mm×100 mm, 1.7 μm) was used for the separation with a mobile phase of water(containing 0.1% diethylamine and 500 ng·mL^-1 guanidine hydrochloride) and acetonitrile(containing 0.1% diethylamine and 500 ng·mL^-1 guanidine hydrochloride) at a flow rate of 0.6 mL·min^-1 in gradient elution mode. The method was developed at ESI(-) with SIR mode. RESULTS The limit of quantitation of tagatoes, fructose, epilactose and lactulose were 0.5, 0.5, 0.2 and 0.2 μg·mL^-1, respectively. The limit of detection of lactose and galactose were 0.2 and 2 μg·mL^-1, respectively. The linearity ranges of the tagatoes, fructose, galactose, epilactose, lactulose, lactose were 5.1-123.2, 4.9-117.8, 5.4-129.8, 5.2-125.0, 5.0-119.9 and 5.0-121.2 μg·mL^-1 respectively, with the excellent correlation coefficient(r ≥ 0.995). The average recoveries were 90%-107% (n=6). The instrument of methods had a good precision. The RSD of peak area were all < 2.8% for the six target compounds(n=5). The contents of lactulose and related substances in lactulose oral solution produced by two different manufacturers were all in accordance with the regulations of Chinese Pharmacopoeia 2015 Edition, and were basically consistent with the results of method in Chinese Pharmacopoeia. CONCLUSION The established method can be applied to the determination of the contents of lactulose and related substances in lactulose oral solution.