While alterations in bone mineral density (BMD) are of interest in a number of musculoskeletal conditions affecting the knee, their analysis is limited by a lack of tools able to take full advantage of modern imaging modalities. This study introduced a new method, combining computed tomography (CT) and computational anatomy algorithms, to produce standardized three-dimensional BMD quantification in the distal femur and proximal tibia. The method was evaluated on ten cadaveric knees CT-scanned twice and processed following three different experimental settings to assess the influence of different scans and operators. The median reliability (intraclass correlation coefficient (ICC)) ranged from 0.96 to 0.99 and the median reproducibility (precision error (RMSSD)) ranged from 3.97 to 10.75 mg/cc for the different experimental settings. In conclusion, this paper presented a method to standardize three-dimensional knee BMD with excellent reliability and adequate reproducibility to be used in research and clinical applications. The perspectives offered by this novel method are further reinforced by the fact it relies on conventional CT scan of the knee. The standardization method introduced in this work is not limited to BMD and could be adapted to quantify other bone parameters in three dimension based on CT images or images acquired using different modalities.