This factorial study examined the potential of slaughtered cattle as a source of rumen fluid (RF) to test the impact of 0, 90, 180 g/kg forage (Amount) of 2 supplements (Low protein of 12% =LP; high protein of 25% =HP) on in vitro rumen degradation of grass nuts (Grass) and barley straw (Straw) over various times. RF was collected from three slaughtered cattle (Animal) on three separate occasions and buffered under CO2 as inoculum at 39°C. About 1g ground Straw or Grass and each amount of LP or HP were mixed in separate test tubes with 50 ml inoculum under CO2. The tubes fitted with Bunsen valves were incubated at 0-72h after which the contents were washed and dried to estimate dry matter disappearance (DMD). The DMD were statistically analysed for the main effects of Animal, Forage, Supplement, Amount and their interactions at each incubation but not Animal based interactions. The Forage, Amount and Animal effects were significant (P<0.01) at most incubations whereas Supplement effect was significant at 72h only showing greater DMD for HP than LP (P<0.05). DMD increased with time for all main effects. Grass had greater DMD than Straw at all incubations (P<0.001). DMD increased (P<0.05) with time but not at 0h with increasing Amount and increased DMD was greater from 0 to 90 g/kg than at 90 to 180 g/kg. The animals differed (P<0.05) at all but 0h showing variable responses with the confounding effect of their gender, age, breed, size and pre-slaughter feeding. Apparently slaughtered cattle can be used to obtain RF to evaluate supplements for in vitro forage degradation. However, the selection of suitable donor animals of similar pre-slaughter characteristics would be critical if in vitro studies involving slaughtered animals are to be used to evaluate feed supplements before their use for grazing ruminants.