Sodium (Na+) ions are known to regulate many signaling pathways involved in both physiological and pathological conditions. In particular, alterations in intracellular concentrations of Na+ and corresponding changes in membrane potential are known to be major actors of cancer progression to metastatic phenotype. Though the functionality of Na+ channels and the corresponding Na+ currents can be investigated using the patch-clamp technique, the latter is rather invasive and a technically difficult method to study intracellular Na+ transients compared to Na+ fluorescence imaging. Despite the fact that Na+ signaling is considered an important controller of cancer progression, only few data using Na+ imaging approaches are available so far, suggesting the persisting challenge within the scientific community. In this study, we describe in detail the approach for application of Na+ imaging technique to measure intracellular Na+ variations in human prostate cancer cells. Accordingly, we used three Na+-specific fluorescent dyes–Na+-binding benzofuran isophthalate (SBFI), CoroNa™ Green (Corona) and Asante NaTRIUM Green-2 (ANG-2). These dyes have been assessed for optimal loading conditions, dissociation constant and working range after different calibration methods, and intracellular Na+ sensitivity, in order to determine which probe can be considered as the most reliable to visualize Na+ fluctuations in vitro.