Background Soil salinity is an abiotic stress wide spread in rice producing areas, limiting both plant growth and yield. The development of salt-tolerant rice requires efficient and high-throughput screening techniques to identify promising lines for salt affected areas. Advances made in image-based phenotyping techniques provide an opportunity to use non-destructive imaging to screen for salinity tolerance traits in a wide range of germplasm in a reliable, quantitative and efficient way. However, the application of image-based phenotyping in the development of salt-tolerant rice remains limited. Results A non-destructive image-based phenotyping protocol to assess salinity tolerance traits of two rice cultivars (IR64 and Fatmawati) has been established in this study. The response of rice to different levels of salt stress was quantified over time based on total shoot area and senescent shoot area, calculated from visible red-green-blue (RGB) and fluorescence images. The response of rice to salt stress (50, 75 and 100 mM NaCl) could be clearly distinguished from the control as indicated by the reduced increase of shoot area. The salt concentrations used had only a small effect on the growth of rice during the initial phase of stress, the shoot Na+ accumulation independent phase termed the ‘osmotic stress’ phase. However, after 20 d of treatment, the shoot area of salt stressed plants was reduced compared with non-stressed plants. This was accompanied by a significant increase in the concentration of Na+ in the shoot. Variation in the senescent area of the cultivars IR64 and Fatmawati in response to a high concentration of Na+ in the shoot indicates variation in tissue tolerance mechanisms between the cultivars. Conclusions Image analysis has the potential to be used for high-throughput screening procedures in the development of salt-tolerant rice. The ability of image analysis to discriminate between the different aspects of salt stress (shoot ion-independent stress and shoot ion dependent stress) makes it a useful tool for genetic and physiological studies to elucidate processes that contribute to salinity tolerance in rice. The technique has the potential for identifying the genetic basis of these mechanisms and assisting in pyramiding different tolerance mechanisms into breeding lines.