The composition of cartilage is predictive of its in vivo performance. Therefore, the ability to assess its primary macromolecular components, proteoglycan (PG) and collagen, is of great importance. In the current study, we hypothesized that PG content and distribution in tissue engineered cartilage could be determined using Fourier-transform infrared imaging spectroscopy (FT–IRIS). The cartilage was grown from chondrocytes within a hollow fiber bioreactor (HFBR) system previously used extensively to study cartilage development. FT–IRIS analysis showed a gradient of PG content, with the highest content in the center near the nutritive fibers and the lowest near the interior surface of the HFBR. Further, we found significantly greater PG content in the region near culture medium inflow (45.0%) as compared to the outflow region (24.7%) (p<0.001). This difference paralleled the biochemically determined glycosaminoglycan difference of 42.6% versus 27.8%. In addition, FT–IRIS-determined PG content at specific positions within the tissue sections correlated with histologically determined PG content (R=0.73, p=0.007). In summary, FT–IRIS determination of PG correlates with histological determination of PG and yields quantitatively similar results to biochemical determination of glycosaminoglycan in developing cartilage.