The ratio of Na+ and K+ is an important determinant of the magnitude of Na+ toxicity and osmotic stress in plant cells. Traditional analytical approaches involve destructive tissue sampling and chemical analysis, where real-time observation of spatio-temporal experiments across genetic or breeding populations is unrealistic. Such an approach can also be very inaccurate and prone to erroneous biological interpretation. Analysis by Hyperspectral Imaging (HSI) is an emerging non-destructive alternative for tracking plant nutrient status in a time-course with higher accuracy and reduced cost for chemical analysis. In this study, the feasibility and predictive power of HSI-based approach for spatio-temporal tracking of Na+ and K+ levels in tissue samples was explored using a panel recombinant inbred line (RIL) of rice (Oryza sativa L.; salt-sensitive IR29 x salt-tolerant Pokkali) with differential activities of the Na+ exclusion mechanism conferred by the SalTol QTL. In this panel of RILs the spectrum of salinity tolerance was represented by FL499 (super-sensitive), FL454 (sensitive), FL478 (tolerant), and FL510 (super-tolerant). Whole-plant image processing pipeline was optimized to generate HSI spectra during salinity stress at EC = 9 dS m-1. Spectral data was used to create models for Na+ and K+ prediction by partial least squares regression (PLSR). Three datasets, i.e., mean image pixel spectra, smoothened version of mean image pixel spectra, and wavelength bands, with wide differences in intensity between control and salinity facilitated the prediction models with high R2. The smoothened and filtered datasets showed significant improvements over the mean image pixel dataset. However, model prediction was not fully consistent with the empirical data. While the outcome of modeling-based prediction showed a great potential for improving the throughput capacity for salinity stress phenotyping, additional technical refinements including tissue-specific measurements is necessary to maximize the accuracy of prediction models.