Glyphosate (GLY) is one of the most commonly used herbicides worldwide. Both GLY and aminomethylphosphonic acid (AMPA), its main degradation product, may be present in feedstuffs offered to dairy cows. Although the major proportions of ingested GLY and AMPA are eliminated with faeces, a potential degradation of GLY to AMPA in the rumen of dairy cows has been suggested. Considering that the rumen plays a central role in the pre-systemic metabolism of xenobiotics, this research aimed to investigate whether or not GLY and AMPA are metabolised in the ruminal environment of cattle. The distribution of both compounds between the fluid and solid phases of the ruminal content (RC) was also evaluated. RC from 3 steers were collected in an abattoir. Aliquots were incubated (3–6 h) in anaerobiosis with GLY (15 µg/mL) and AMPA (1.5 µg/mL). Metabolic viability of RC was assessed by the measurement of the sulpho-reduction of the anthelmintic derivative albendazole sulphoxide (ABZSO) into albendazole (ABZ) in the absence (controls) or in presence of GLY and AMPA. Incubations of boiled (inactive) RC were used as controls. Samples were analysed by HLPC with fluorescence detection. Neither GLY nor AMPA were metabolised in metabolically active RC from cattle. Both compounds were predominantly found in the fluid phase compared to the solid (particulate) matter of RC. Neither GLY nor AMPA had a negative effect on the metabolic production of ABZ. A high metabolic stability of both compounds within the ruminal environment would be expected in vivo. Their presence in high proportion in the fluid phase of the ruminal content may give rise to a rapid flow of both GLY and AMPA to the posterior gastrointestinal tract. Negative effects on the ruminal biotransformation of therapeutically used drugs would not be expected when the herbicide and its degradation product are consumed with food.