The collagen network is the highly organized backbone of articular cartilage providing tissue tensile stiffness and restricting proteoglycan bleaching out of the tissue. Osteoarthritis (OA) diminishes proper collagen network adaptation. Our aim was to provide quantitative three-dimensional (3D) information of the cartilage collagen network adaptation in early osteoarthritis using high resolution micro-computed tomography (µCT)-imaging. Osteochondral samples from the femoral condyles were collected from healthy (N= 8, both legs) and experimental OA rabbit model with anterior cruciate ligament transection (N= 14, single leg). Samples were processed for cartilageµCT-imaging and histological evaluation with polarized light microscopy (PLM). Structure tensor analysis was used to analyse the collagen fibre orientation and anisotropy of theµCT-images, and PLM was used as a validation for structural changes. Depth-wise comparison of collagen fibre orientation acquired withµCT-imaging and PLM correlated well, but the values obtained with PLM were systematically greater than those measured withµCT-imaging. Structure tensor analysis allowed for 3D quantification of collagen network anisotropy. Finally,µCT-imaging revealed only minor differences between the control and experimental groups.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10439-023-03183-4.