To investigate deeper microscopic information of articular cartilage and subchondral bone components in situ, Raman spectroscopy combined with tissue optical clearing technique was used to study the clearing effect of articular cartilage. Raman spectra of the cartilage adhered on femur ends in canine knees were collected after they were impregnated at different time and different concentrations of clearing agents, glycerol and iohexol. The integrated Raman intensity ratio of phosphate group (920~960 cm-1) to the amide Ⅰ band (1 595~1 700 cm-1) was obtained to seek the rule of tissue optical clearing at different impregnating time (10～60 min) and different concentrations of clearing agents (40%, 60%, 80% and 100% for glycerol, 50, 150, 250, 350 mg·mL-1 for iohexol), which is to get the optimum concentration and time. It was found that the both agents could enhance the Raman signal compared to the case of no clearing agent. With equal time for impregnating, glycerol and iohexol were able to obtain good results at 60% and 150 mg·mL-1, respectively. Under different concentrations, the clearing effect of glycerol was the strongest in 20 min, while that of iohexol was enhanced after 50-min impregnation in general. In conclusion, the Raman scattering information of subchondral bone can be directly detected when the clearing agents penetrating through cartilage, which will provide a novel way and a potential solution into study on osteoarthritis etiology in depth.