The aim was to evaluate the effect of replacing maize silage (MS) plus soybean meal with red clover silage (RCS) plus wheat grain in total mixed rations (TMR) on utilizable crude protein at the duodenum (uCP), and to evaluate the modified Hohenheim gas test (moHGT) for estimation of uCP supply in dairy cows. The TMR comprised forage and concentrates (0.75:0.25), and the forage portion comprised of MS and RCS. The RCS accounted for 0.15, 0.30, 0.45, or 0.60 of the TMR dry matter. The uCP of the TMR was calculated using the "standard German procedure" as the reference value (uCP(GfE)) as the sum of ruminally undegraded feed crude protein (RUPGfE) and ruminally synthesized microbial crude protein (MCPGfE). For this, the RUPGfE content was calculated from RUP content based on in situ ruminal degradation of crude protein, and the MCPGfE was calculated based on the contents of crude protein, RUP and metabolizable energy. The moHGT was used to determine the in vitro uCP (uCP(moHGT)) and was compared with uCP(GfE). The RUPGfE, MCPGfE, uCP(GfE) and uCP(moHGT) were estimated assuming passage rates out of the rumen of 2, 5 and 8%/h. At all assumed passage rates, replacing MS with RCS linearly decreased (P < 0.001) the RUPGfE, MCFGfE and uCP(GfE) content of the TMR. The uCP(moHGT) at passage rates of 5 and 8%/h was closely correlated with uCP(GfE) (R-2 = 0.94, P = 0.03, and R-2 = 0.95, P = 0.02, respectively), whereas for a passage rate of 2%/h there was no significant correlation (R-2 = 0.72, P = 0.15). In conclusion, increasing the proportion of RCS plus wheat grain in the diet of dairy cows reduced the amount of uCP(GfE) and this was caused more by a reduction in the RUPGfE than in the MCPGfE content. The moHGT has considerable potential as an accurate method for estimating the uCP of TMR, but further studies on standardization of the method and validation with a greater number of TMR samples are recommended.