Introduction: Subchondral bone mineral density (sBMD) contributes to the initiation and progression of knee osteoarthritis (OA). Reliable methods to assess sBMD status may predict the response of specific OA phenotypes to targeted therapies. While dual-energy X-ray absorptiometry (DXA) of the knee can determine sBMD, no consensus exists regarding its methodology. Objective: Construct a semi-standardized protocol for knee DXA to measure sBMD in patients with OA of the knee by evaluating the varying methodologies present in existing literature. Methods: We performed a systematic review of original papers published in PubMed and Web of Science from their inception to July 2014 using the following search terms: subchondral bone, osteoarthritis, and bone mineral density. Results: DXA of the knee can be performed with similar reproducibility values to those proposed by the International Society for Clinical Densitometry for the hip and spine. We identified acquisition view, hip rotation, knee positioning and stabilization, ROI location and definition, and the type of analysis software as important sources of variation. A proposed knee DXA protocol was constructed taking into consideration the results of the review. Conclusions: DXA of the knee can be reliably performed in patients with knee OA. Nevertheless, we found substantial methodological variation across previous studies. Methodological standardization may provide a foundation from which to establish DXA of the knee as a valid tool for identification of SB changes and as an outcome measure in clinical trials of disease modifying osteoarthritic drugs.