The objectives of the study were to examine the effect of an antibiotic solution applied in the Streptomyces griseus protease method (SGPM) and the effect of carbohydrases in SGPM on the effective crude protein (CP) degradation (ED) with reference to in sacco ED. For this purpose, the ruminal CP degradation of rapeseed meal, dried distillers’ grains with solubles, wheat grain, corn grain, corn silage, grass silage and partial crop field pea silage was determined in sacco using three rumen-fistulated dairy cows and in vitro using SGPM. The impact of the antibiotic solution on CP degradation by S. griseus protease was investigated by supplementing SGPM with Penicillin–Streptomycin solution to reduce microbial mass proliferation during incubation. The carbohydrase α-amylase or Viscozym® L (cell wall-degrading enzyme mixture) was added to the SGPM at four different doses simultaneously as a co-incubation to improve feed protein accessibility. For most feedstuffs, ED was lower when the antibiotic solution was used in SGPM (p p S. griseus protease and carbohydrase was not successful in reducing the differences to the in sacco CP degradation.