The aim of this work is to demonstrate the presence of proline in both root tissues and root exudates as a response of excised root cultures to salt stress and to ascertain the possible relationship between proline content and NaCl concentration. Roots from micropropagated Prunus rootstocks have been cultured in vitro under increasing NaCl concentrations (0, 20, 60 and 180 mM) to early detect their tolerance to salt stress. After 3 weeks of culture, the proline content of the MS-based liquid medium, in which roots were cultured, was determined. As far as we know, no attempts have been made to determine the proline content of excised root cultures and root exudates under stress. Proline concentration in root tissues and root exudates from all rootstocks increased as salt concentration in the medium increased, following a trend similar to that of whole plant tissues in all three Prunus rootstocks (P. insititia, ‘Adesoto 101’; P. cerasus, ‘Masto de Montañana’ and P. dulcis x persica, GF 677). This opens the possible role of proline exudates to study plant responses to salt stress using nondestructive methods. In addition, proline exudates can be of great interest in the early detection of salt stress tolerance, provided that a relation between proline and salt stress tolerance could be found.