Lameness due to femoral head separation (FHS) is a production and welfare issue in commercial poultry. FHS is an idiopathic disorder, which is attributed to a myriad of factors but in order to improve bone health, broiler breeders must be prognosed for disease susceptibility and selected against FHS using biomarkers. Proteins from plasma of blood, which can be obtained using minimally invasive methods represent an ideal, rich source of biomarkers which might be different in susceptible or affected birds. The peptide and proteins in plasma of healthy (HLTH) and affected birds (FHS) were compared using Matrix assisted laser desorption ionization mass spectrometry (MALDI-TOF-MS) and Liquid chromatography and tandem mass spectrometry (LC-MS/MS). The peptide profile of HLTH and FHS were compared using ClinPro tools and the differentially expressed peptides were isolated by Reverse phase liquid chromatography fractionation (C18-RP-HPLC) and identified using peptide mass fingerprinting. Peptides derived from fibrinogen precursor and fetuin were reduced in FHS birds. Based on the proteomic analysis, proteins such as Gallinacin 10, Apolipoprotein A-1 and Hemoglobin chains are elevated in FHS while Alpha 1-acid glycoprotein is reduced in FHS birds. Our study shows that bodyweight, lipid profile and the above mentioned proteins could be useful as a biomarker for improvement of bone health. These proteins indicate that blood lysis, antimicrobial defense and lipid disorder but lack of an inflammatory response might be consequential to FHS.