The purpose of this study was twofold. Firstly, we proposed a measurement protocol for the atomic force microscopy (AFM) method to determine the nanomechanical properties of articular cartilage in experimental osteoarthritis in rabbits. Then, we verified if mechanical properties can be evaluated with AFM shortly after platelet-rich plasma (PRP) injection. We hypothesized that the modulus determined by AFM indentation experiments could be utilized as a progressive disease marker during the treatment of osteoarthritis. The rabbits were equally divided into three groups of six: control (group 1); injections of saline (0.5 mL) and 10% surgical talc (Talcum Pharmaceutical®, Minsk, Belarus) were delivered into the right knee under the patella (group 2 and 3); and PRP was injected into the right knee (group 3). In group 2, the arithmetic average of absolute values (Ra) change was a 25% increase; the maximum peak height (Rp) increased by over 102%, while the mean spacing between local peaks (S) increased by 28% (p < 0.05). In group 3, Ra increased by 14% and Rp increased by 32%, while S decreased by 75% (p < 0.05). The Young’s modulus of the surface layers decreased by 18% as a result of induced model of osteoarthritis (IMO) (p < 0.05), and it increased by 9% (p < 0.05) as a result of PRP therapy, which means that the mechanical properties of cartilage were partially recovered. This research demonstrates that Young’s modulus utilized on a nanometer scale has potential to be a progressive disease marker during the treatment of osteoarthritis.