An in vitro study was conducted to determine the extent of melamine degradation in rumen liquor. Rumen liquor was collected from two ruminally cannulated Holstein cows on four separate dates, one week apart. Erlenmeyer flasks (250 mL) were prepared for incubation by adding 1000 mg of a dairy feed substrate, 100 mg melamine and 100 mL incubation medium, purged with CO2 and fitted with rubber stoppers equipped with one-way gas release valves. The initial melamine concentration was thus 1000 mg/L. The substrates consisted of 600 mg of a commercial dairy concentrate, 200 mg lucerne hay and 200 mg oat hay. The incubation medium consisted of 19 mL rumen liquor, 77 mL of Van Soest buffer and 4 mL of a reducing solution. The flasks were incubated at 39 ºC for 0, 6, 24 or 48 hours (two flasks per time in each of four replicates). The 0 h incubation served as a control treatment to enable the calculation of melamine recovery values. For the control treatment (0 h), fermentation was terminated at the onset of the trial by aerating the rumen liquor and submerging the flasks in 50 mm ice. On termination of the incubation, 100 mL 0.2 M perchloric acid was added to each flask in order to dissolve any undegraded melamine. Melamine concentrations were determined by liquid chromatography-tandem mass spectrometry. Melamine degradation was low after 6 hours and 24 hours of incubation (3.2% and 5.5%, respectively) and increased to 13.6% after 48 h of incubation. It was concluded that melamine has low degradability in rumen liquor.Keywords: Non-protein Nitrogen, Rumen Fermentation, Rumen Incubation