In situ estimates of ruminal undegraded fraction (RU) and effective intestinal digestibility (EID, corrected for microbial colonisation) of dry matter (DM), crude protein (CP) and total analysed amino acids (TAA) of rye, wheat and corn grains, wheat bran, wheat and barley distillers’ dried grains with solubles (DDGS) and corn gluten feed were measured on three rumen and duodenum cannulated wethers using 15N labelling techniques and considering ruminal rates of particle comminution (kc) and outflow. Results indicate that not considering kc and microbial colonisation led to considerable overestimations of RU which increased with feed ruminal degradation. Microbial colonisation may be also associated with overestimations of EID, whose estimates for DM, CP and TAA were predicted from parameters related with the ruminal escape of intestinally indigestible materials. The RU estimates were higher for TAA than for CP in grains, but the opposite was observed in by-products, whereas EID estimates were higher for TAA in all feeds. To obtain accurate protein values in these feedstuffs, it is required to consider both kc and ruminal microbial colonisation. The CP-based results underestimate the intestinally digested protein in grains and the opposite is evidenced in cereal by-products. Microbial protein synthesised in the rumen is largely the major fraction of the feedstuff protein value with the exception of DDGS.