This study addressed difficulties in evaluating osteoarthritis (OA) progression in species with thin cartilage. Feasibility of using short, nonequilibrium contrast-enhanced micro-computed tomography (CE-μCT) to evaluate the physical and biochemical properties of cartilage was investigated. A preliminary in vitro study using CE-μCT study was performed using bovine osteochondral blocks with intact, mildly damaged (fibrillated), or severely damaged (delaminated) cartilage. Delamination of the superficial zone resulted in elevated apparent density compared with intact cartilage after 10 minutes of anionic contrast exposure (P < 0.01). OA was induced by unilateral meniscal destabilization in n = 20 sheep divided into: early phase OA (n = 9) and late phase OA (n = 11), while n = 4 remained as naive controls. In vivo anionic nonequilibrium contrast CT of the operated stifle was conducted in the early phase sheep 13 weeks postoperatively using clinical resolution CT. Cartilage visibility in the contrasted leg was significantly improved compared with the noncontrasted contralateral stifle (P < 0.05). Animals were sacrificed at 3 months (early phase) or 12 months (late phase) for additional ex vivo CE-μCT, and correlative tests with biochemical and histological measures. Concentration of sulfated glycosaminoglycan (sGAG) significantly varied between control, early, and late phase OA (P < 0.005) and showed a negative (r = -0.56) relationship with apparent density in the medial tibial plateau (R2 = 0.28, P < 0.001). Histologically, parameters in proteoglycan and cartilage surface structure correlated with increasing attenuation. While previous studies have shown that CE-CT increases the apparent density of proteoglycan-depleted cartilage, we concluded that superficial zone disruption also contributes to this phenomenon.