Spent hens have low market value and incur significant costs for disposal. This study explored the use of spent hens as a source of bioactive peptides. Spent hen hydrolysates prepared by Protease M or Protex 50FP exhibited interleukin (IL)-6 inhibitory activity in endotoxin-activated macrophage-like U937 cells (p < .05). The potential peptides from Protex 50FP hydrolysate were further fractionated using a combination of ultrafiltration, solid-phase extraction and high-performance liquid chromatography; 17 novel peptides encoded in major muscle proteins were identified by mass spectrometry analysis, of which 7 were chemically synthesized and assayed for the IL-6 inhibitory activity. At a concentration of 100 µg/mL, peptide FLWGKSY induced a 79% reduction of IL-6 production in endotoxin-activated macrophage-like U937 cells, which is comparable to results reported from other food sources. Our results indicate that spent hens have potential to be a source of bioactive peptides for anti-inflammatory applications.