Nutritive value of alfalfa hay is influenced by stage of maturity and cutting time which affects the potential nutrient supply in dairy cows. The objective of this study was to investigate the effect of stage of maturity and cutting time of alfalfa hay grown under semi-arid climate condition on in situ ruminal degradation characteristics and predicted protein availability in dairy cows using two protein models based on different principles (DVE/OEB 1994 and NRC, 2001). Alfalfa was cut at early bud (June 15/16), late bud (June 26/27) and early flower (July 18/19) in the afternoon (18:00 h) and the following morning (06:00 h). Alfalfa hay at early bud and late bud contained higher in situ effective degradable nitrogen (EDN) to effective degradable energy (ED organic matter (EDOM) and ED carbohydrates (EDCHO)) ratios compared with alfalfa at early flower (P<0.05) and highest EDN to EDOM and EDCHO ratios were reached in the first hours of ruminal incubation for all alfalfa hays. Rumen degraded protein balance decreased with advancing maturity (P<0.05). There was a trend towards reduction in metabolizable protein (NRC model; 78, 72 and 66 g/kg dry matter (DM), P=0.06) and truly absorbed protein (DVE value; 57, 51 and 39 g/kg DM, P=0.08) with advancing maturity. Cutting alfalfa in the afternoon increased EDCHO (450 vs. 431 g/kg CHO; P=0.08), ruminal microbial protein synthesis based on total digestible nutrients (NRC model; MCPE NRC; 62 vs. 59 g/kg DM; P=0.03) and intestinally absorbable MCPE NRC (40 vs. 38 g/kg DM; P=0.03) compared with cutting alfalfa in the morning. In conclusion, cutting alfalfa hay at early bud stage tended to have the highest metabolizable protein content, but also the highest imbalance between rumen available protein and energy and cutting alfalfa in the afternoon increased potential ruminal microbial protein synthesis in dairy cows.