When formulating dairy cow rations, characterization of protein in feeds requires estimation of protein degradation in both the rumen and intestine. The objective of this work was to evaluate experimental and feed related factors that affect characterization, using in situ, in vitro, or mobile bag techniques, of 0-h washout (A), potentially degradable (B), and undegradable (C) protein fractions, protein degradation rate (K_d), and digestibility of rumen undegradable protein (dRUP). Data sets of 136 studies on A, B, C, and K_d and 113 studies on dRUP were amassed from the literature. Mixed-effect linear models were used to relate these variables to methodological and feed factors while accounting for random differences among studies. Predictions of A, B, and C protein fractions were significantly (P < 0.05) influenced by CP and NDF interactions with sample grind size, bag pore size, incubation time, bag area, and sample-to-bag area ratio. For example, a 20% decrease in CP of a theoretical feed sample would increase A fraction prediction by 16.7%, but only 8.69% with bag pore size −1 SD below the mean. A shift in measurement method halves the predicted A fraction of the same feed. Similarly, reported K_d values were significantly (P < 0.05) influenced by CP interactions with sample grind size, bag area, and sample-to-bag area ratio. Feed variables and measurement variables influencing protein digestibility measures suggest that these analytical factors are likely associated with variance among differing methodologies and within unique samples of the same feed. When predicting dRUP, pepsin-acid incubation time and use of mobile bag method produced significantly different (P < 0.05) estimates compared with the traditional in vitro 3-step method. The use of mobile bag resulted in a 12% (±3.1%) higher estimate of dRUP compared with the in situ technique. In 618 and 977 samples, sample variation to sample mean ratio for ADF and pepsin-acid incubation time was 63% and 58%, respectively. Variation in feedstuff content and lack of standardization of methods used to measure protein disappearance lead to a lack of robustness in the measurements commonly employed.