Pineapple waste silage (PAS) is an abundant agro-industrial by-product characterized by its high fiber content posing a high potential feed value as roughage for ruminants. Studies on its supplementation with proteolytic enzyme (PE) will help extend its utilization as an alternative nutritive feed source for cattle nutrition. Thus, this study aimed to determine the in vitro nutrient degradability and fermentation characteristics of fiber-rich but low-protein PAS supplemented with different levels of PE. Seven treatments were evaluated in this study: PAS without PE and PAS1 to PAS6, which corresponds to incremental levels of PAS supplementation as follows: 0.1%, 0.2%, 0.3%, 1%, 2%, and 4%. The nutrient disappearance, nutrient effective degradability, and fermentation characteristics such as total gas production, ammonia-nitrogen, and pH values were evaluated in vitro. PAS without added PE showed a comparably good nutritive value (dry matter: 94.30%, neutral detergent fiber: 63.66%, acid detergent fiber: 34.78%) to that of commonly used corn silage in South Korea. With the supplementation of PE in PAS, the PE increased the effective degradability of different nutrients such as dry matter (DM), neutral detergent fiber (NDF), acid detergent fiber (ADF), organic matter (OM), and crude protein (CP). The effect of PE supplementation on the degradation of nutrients was consistent with improvements in in vitro rumen fermentation characteristics. Supplementing PAS with PE increased the total gas production and decreased the pH values, which are characteristics of heightened fiber degradation and fermentation. The ammonia-N concentration of the in vitro-incubated PAS was moderated by the addition of PE, which is likely due to the decrease in pH or in vitro acidosis and has shown a synergistic protease activity effect on nutrient degradation. Overall, supplementing PAS with PE increased the effective degradability of DM, NDF, ADF, OM, and CP, with the most dramatic effects observed in PAS3 and PAS6 (0.3% and 4%, respectively).