This study was conducted to determine the effect of different vegetative periods on protein fractions of alfalfa hay and to compare two different methods for estimation of its protein degradability. In this study, 44 alfalfa hay samples cut in late vegetative, late bud, early bloom and late bloom were used. Crude protein decreased with advancing maturity (p <.05), but neutral detergent insoluble nitrogen and acid detergent insoluble nitrogen were similar. Protein degradability of the samples was estimated using Streptomyces griseus protease or borate–phosphate buffer, kinetic model, as described in Cornell Net Carbohydrate Protein System (CNCPS). Alfalfa hay samples were subjected to proteolysis for 30 h with 0.115 U/mL or 0.230 U/mL of the protease at pH 6.8. There was a moderate correlation (0.66, 0.72) between enzymatic and buffer method estimates with protein degradation. As vegetation progresses, rapidly degradable fraction (A) decreased (p <.05) and undegradable fraction (C) increased (p <.05), while potentially degradable fraction (B) was unchanged. Rumen degradable protein content decreased (p <.05) with advancing maturity. The enzyme/duration limits should be standardised and investigated whether the protease enzyme alone is sufficient for all forages.