Osteoarthritis is associated with changes to the matrix composition of subchondral bone. Raman spectroscopy has the potential to detect in vivo the molecular changes in osteoarthritic subchondral bone. The objectives were to determine the levels of mineralisation, carbonate accumulation and bone remodelling in osteoarthritic subchondral bone, which we defined as within 3mm of articular cartilage. This was compared to the proximal-compartment (10mm distal to articular cartilage) and the head-neck junction. Five osteoarthritic (average age: 76 years) and five normal cadaveric femoral heads (average age: 72 years) were scanned using peripheral quantitative computed tomography and then sectioned coronally. Raman spectroscopy was then used to scan the femoral heads. All scans were done in the plane of the longitudinal axis of the diaphysis. Cores were subsequently extracted and sodium dodecyl sulphate polyacrylamide gel electrophoresis performed to determine the levels of homotrimeric collagen. The phosphate-to-amide I ratio, from the Raman spectra, in osteoarthritic subchondral bone was significantly greater than controls (p=0.023). Within osteoarthritic specimens, the phosphate-to-amide I ratio increased proximally. The density in osteoarthritic subchondral bone was 89mg/cm3 higher than controls (p=0.022), and 494mg/cm3 higher than the osteoarthritic proximal-compartment (p<0.001). Moreover, carbonate substitution into the apatite crystals decreased in osteoarthritic specimens. The carbonate-to-amide I ratio was highest in osteoarthritic subchondral bone. Furthermore, the median α1-to-α2-chain ratio in osteoarthritic specimens was 2:1. The changes found in subchondral bone are important in the pathogenesis of osteoarthritis. This study shows that Raman spectroscopy can detect differences between osteoarthritic specimens and controls, further supporting its potential use in diagnosing bone disorders.