Alfalfa (Medicago sativa L.) has high crude protein that is rapidly and extensively degraded in the rumen. Our objective was to develop a protocol where individual proteins could be characterized for their ruminal degradation. Proteins from individual genotypes of three alfalfa cultivars were characterized using fluorescence 2D difference gel electrophoresis combined with MALDI-TOF mass spectrometry for protein identification. Twenty-six proteins were characterized, representing between 33 and 41% of the total protein among genotypes. Variation for protein degradation was observed among proteins after 45 and 120 min of incubation in the rumen of a Holstein steer (P < 0.001). After 45 min of ruminal incubation, nine proteins averaged 75% or more remaining, 12 had 50% or less remaining, and five were intermediate. After 120 min of ruminal incubation, four proteins averaged greater than 80%, seven between 80 and 50%, and 15 less than 50% remaining. Although all proteins were degraded over time, the rate and amount of degradation was dramatically different among them. The rate of digestion differed (P = 0.05) for 3 and 10 proteins among genotypes after 45 and 120 min, respectively. Individual proteins characterized ranged in mass from 41 to 0.29% of the total mass of protein characterized. Total content of those proteins that differed for rate of digestion ranged from 7 to 1%. The results demonstrate that individual proteins can be characterized for their ruminal degradation. The ability to separate proteins based ruminal degradation suggests there is potential to select for protein that degrades more slowly and possibly escapes the rumen.Key words: Alfalfa, protein, rumen, digestion