In diseased conditions of cartilage such as osteoarthritis, there is typically an increase in water content from the average normal of 60–85% to greater than 90%. As cartilage has very little capability for self-repair, methods of early detection of degeneration are required, and assessment of water could prove to be a useful diagnostic method. Current assessment methods are either destructive, time consuming or have limited sensitivity. Here, we investigated the hypotheses that non-destructive near infrared spectroscopy (NIRS) of articular cartilage can be used to differentiate between free and bound water, and to quantitatively assess water content. The absorbances centered at 5200 cm−1 and 6890 cm−1 were attributed to a combination of free and bound water, and to free water only, respectively. The integrated areas of both absorbance bands were found to correlate linearly with the absolute water content (R=0.87 and R= 0.86) and with percent water content (R=0.97 and R=0.96) of the tissue. Partial least square models were also successfully developed and were used to predict water content, and percent free water. These data demonstrate that NIRS can be utilized to quantitatively determine water content in articular cartilage, and may aid in early detection of degenerative tissue changes in a laboratory setting, and with additional validations, possibly in a clinical setting.