Beta-endorphin 1-31 (BE) is an endogenous opioid peptide previously demonstrated to play roles in pain, reward, stress and the immune system. During inflammation, immune cell expression, production, and the release of BE increases within inflamed tissue. However, within the inflamed milieu BE is also susceptible to increased enzymatic degradation. BE and its three fragments were incubated in inflamed tissue homogenates at pH 5.5 for 2 hrs. The supernatants were separated using liquid chromatography/ mass spectrometry (LC/MS) on a C4 column followed by peak detection and identification using electrospray mass spectrometry on an API 3000 tandem mass spectrometer. BE 1-13 and BE 1-11 were identified as two of the major metabolites after incubation of BE 1-31 and BE 1-17 in inflamed tissue homogenates. BE 2-9, BE 2-13, and BE 2-17 were similarly the major metabolites after incubation of BE 1-17; BE 1-11 and BE 2-13 were detected in early fractions of BE 1-13 following incubation. The fragments, 2-11, 3-11, 4-11 and 2-9 were detected following incubation of BE 1-11. Potential hydrolysis sites of BE 1-31 were detected between the following amino acids: Lys⁹-Ser¹⁰, Pro¹³-Leu¹⁴, Leu¹⁷-Phe¹⁸, Phe¹⁸-Lys¹⁹, Lys¹⁹-Asn²⁰. The primary site of hydrolysis in BE 1-17, 1-13, and BE 1-11 was shown to be between amino acids Tyr¹-Gly², providing analogues typically reported to be devoid of opioid activity.