Chondroitin sulfate (CS) is an example of a type of acidic mucopolysaccharides, which consist of repeating disaccharide units of glucuronic acid and galactosamine. Since CS has no UV chromophore it is usually detected by its terminal absorption at a wavelength of 200 nm due to the N-acetyl function, resulting in lower sensitivity. Raman spectroscopy (Raman) and near-infrared spectroscopy (NIR) coupled with partial least squares (PLS) can provide rapid, simple, reproducible and non-destructive quantitative analysis of CS, and no sample pre-treatment or pre-separation are required. In the present study we predicted the CS content of tablets using Raman and NIR combined with PLS. Our results showed that the predicted values obtained by NIR were in good agreement with the actual values, with a correlation coefficient of 0.994. In Raman spectroscopy studies, when the CS content of tablets was within the range 7–39% the correlation coefficient and root mean square error of calibration (RMSEC) were 0.998 and 0.578, respectively. When the CS content of tablets was within the range 41–67% the correlation coefficient and RMSEC were 0.994 and 0.742, respectively. High accuracy could thus be achieved within a wider concentration range by use of NIR, whereas relatively high accuracy could be obtained only within a limited concentration range by the use of Raman spectroscopy.