The objective was to test the hypothesis that ruminal inoculum obtained from slaughtered cattle can replace inoculum from cannulated cattle in trials evaluating animal feedstuffs through in vitro gas production and digestibility. Five adult Holstein × Zebu steers with ruminal cannula were used to collect and compare rumen liquid from in vivo and slaughtered animals. In vitro dry matter digestibility (IVDMD), in vitro neutral detergent fiber digestibility (IVNDFD) and ammoniacal nitrogen concentrations did not differ between inoculum sources (slaughtered × cannulated) for all byproducts and levels tested. Total in vitro gas production in the ruminal inoculum of cannulated animals was greater (P < 0.001) than slaughtered cattle for different levels of licuri cake. However, the greatest total concentrations of in vitro gases for slaughtered animals were observed when evaluating different levels of crude glycerin (P < 0.001). No differences were observed for diets containing castor bean meal (P >0.05). Thus, the ruminal inoculum obtained from the ruminal contents of slaughtered cattle can replace the use of fistulated animals and is a viable alternative to digestibility analysis. This approach is ethically more correct because it alleviates the suffering of animals by avoiding an invasive procedure.