Accumulation of proline in higher plants is an indication of disturbed physiological condition, triggered by biotic or abiotic stress condition. Free proline content can increase upon exposure of plants to drought, salinity, cold, heavy metals, or certain pathogens. Determination of free proline levels is a useful assay to monitor physiological status and to assess stress tolerance of higher plants. Here we describe three methods suitable for determination of free proline content. The isatin paper assay is simple and is suitable to assay proline content in large number of samples. The colorimetric measurement is quantitative and provides reliable data about proline content. The HPLC-based amino acid analysis can be employed when concentration of all amino acids has to be compared.