The present research was performed to investigate endogenous release of bioactive peptides in beef during postmortem aging times (1, 10 and 20 days). Gradually decreased Warner-Bratzler shear force (WBSF) values of longissimus thoracis (LT) and semitendinosus (ST) muscles were observed and the degradation of structural proteins and collagen led to release of low-molecular weight (< 3 kDa) peptides. These peptides exhibited 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity, ACE- and renin-inhibitory activities. The peptide sequences were identified by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). In silico analysis (PeptideRanker and BIOPEP) of their bioactivity potentials demonstrated peptides with the predicted bioactivity scores (> 0.8) as well as collagen peptides with bioactivity scores (0.6–0.8). The present findings provide insights on development of healthy beef through postmortem aging at 4 °C.