Abstract A major objective in nutritional management of high yielding dairy cows is to reduce degradation of dietary protein by rumen microorganisms. The plant kingdom offers a huge diversity of complex compounds that may modify rumen proteolysis. To screen large sample collections and identify plants containing antiproteolytic compounds, rapid and efficient methods are required. Many assays to examine antiproteolytic effects are short term and address direct interferences with protease activities, but isolate the process of proteolysis from overall fermentation. This study aimed to develop a screening system based on a compound substrate, and adequate incubation times, to determine effects on carbohydrate fermentation and microbial growth as well. We describe a simple approach based on batch incubations with rumen fluid to monitor protein degradation over a 12 h incubation. To optimise the system, particular attention was given to the choice of the protein source in the mixed substrate. Casein, bovine serum albumin (BSA), RubisCo and soy protein were assessed with respect to solubility in the buffer medium, a well as detectability of single protein bands on polyacrylamide gels and degradation rates. The ionophore monensin was used as a positive control, due to its known efficacy, and effects were compared to those from in vitro and in vivo studies to validate results from our screening system. The rapid degradation of casein by rumen proteases impeded detection of monensin effects on proteolysis, thereby demonstrating that protein quality crucially impacts efficacy of additives in the assay. Finally, a mixture of soy protein and BSA at a total concentration of 169 g protein/kg was chosen as the standard. Reported effects of monensin on proteolysis and fibre degradation were demonstrated in the assay in a dose dependent manner and 3 μM monensin was chosen for routine application. The system has the advantage of distinguishing between substrate and microbial protein by a centrifugation step and directly monitoring disappearance of substrate protein or single protein bands. Concomitant measurement of gas production, SCFA, and proteolytic end products – ammonia and branched SCFA – provides additional information on overall fermentation as well as on amino acid deamination. The resulting assay was successfully applied for screening in the EC Framework V project, ‘Rumen-up’ to select antiproteolytic plants.