The objective of the current study was to compare the rumen inoculum of slaughtered cattle with the ruminal inoculum of cannulated cattle; additionally, determine its reliability in the in vitro analysis of ruminant feeds throughout a multivariate approach. Five male bovines (weight 320 ± 9.4 kg; mean ± standard deviation) provided with ruminal cannula and between five and seven bovines slaughtered in slaughterhouse were used. The evaluations were carried out following a completely randomized design. The data obtained were subjected to different multivariate analyzes to determine the reliability of the ruminal inoculum of animals slaughtered in commercial slaughterhouses compared to that obtained from cannulated animals. The relative contribution indicated that the in vitro dry matter digestibility (IVDMD, 50.75%) and in vitro neutral detergent fiber digestibility (IVNDFD, 29.83%) analysis influence 80.13% of the results to determine the reliability of the ruminal inoculum from slaughtered cattle. Furthermore, it was determined that the first two principal components (IVNDFD and acetic acid production) are the ones that influence the results by 89.87%. The grouping of diets using the Tocher optimization method and the dendrogram shows the formation of six groups and two groups, respectively. The grouping shows that the ruminal inoculum source was not the limiting parameter in the evaluation. Rumen inoculum from cattle slaughtered in a commercial slaughterhouse (with unknown diet) has potential as an alternative for the in vitro analysis of cattle feed, provided that the lignin concentration in the diet is less than 35.5 g/kg DM.