The spotted hyena (Crocuta crocuta) was an important large carnivore of Pleistocene ecosystems in Africa and Eurasia. Like its modern relatives, this obligate carnivore was adapted to crush and digest bones of its prey and absorb organic matter from bones more efficiently than any other carnivore. This difference in the nutrient resource use between hyenas and most other carnivores led to differences in the isotope flux and variation in the carbon and nitrogen isotopic composition. In our paper, we assess the prey-to-hyena collagen-to-collagen Δ13C and Δ15N trophic discrimination factor (TDF), a key parameter needed in mixing models used for quantitative reconstruction of diet. We analyzed a Pleistocene hyena den bone accumulation in Perspektywiczna Cave (Poland), with a preserved assemblage of remains containing both hyenas and a wide spectrum of their prey represented by digested bones. With the use of proteomics-based taxonomic identification (ZooMS), we estimated the proportion of prey species in the hyena diet. The modeled collagen-to-collagen TDFs are around +1.6‰ to +1.7‰ for δ13C and around +3.4‰ to +3.5‰ for δ15N. This study provides new data on the dietary habits of this large carnivore and allows for a more accurate use of isotopic signals in modeling past hyena diets.