This research aimed to assess the feasibility of utilizing Raman spectroscopy in plant breeding programs. For this purpose, the evaluation of the mutant populations set up the application of 4 mM NaN3 to the somatic embryos obtained from mature wheat (Triticum aestivum L. Adana-99 cv.) embryos. Advanced wheat mutant lines, which were brought up to the seventh generation with salt stress tolerance by following in vitro and in vivo environments constructed by mutated populations, were evaluated using conventional techniques [measurement of antioxidant enzyme activities (SOD, CAT, and POX), total chlorophyll, TBARS, and proline contents; measurement of the concentration of Na+ and K+ ions; and evaluation of gene expression by qPCR (TaHKT2;1, TaHKT1;5, TaSOS1, TaNa+/H+ vacuolar antiporter, TaV-PPase, TaV-ATPase, and TaP5CS)] and Raman spectroscopy. In this research, no significant difference was found in the increase of SOD, CAT, and POX antioxidant enzyme activities between the salt-treated and untreated experimental groups of the commercial cultivar, while there was a statistically significant increase in salt-treated advanced generation mutant lines as compared to control and the salt-treated commercial cultivar. Proline showed a statistically significant increase in all experimental groups compared to the untreated commercial cultivar. The degradation in the amount of chlorophyll was lower in the salt-treated advanced generation mutant lines than in the salt-treated commercial cultivar. According to gene expression studies, there were statistical differences at various levels in terms of Na+ and/or K+ uptake from soil to plant (TaHKT2;1, TaHKT1;5, and TaSOS1), and Na+ compartmentalizes into the cell vacuole (TaNa+/H+ vacuolar antiporter, Ta vacuolar pyrophosphatase, and Ta vacuolar H+-ATPase). The expression activity of TaP5CS, which is responsible for the transcription of proline, is similar to the content of proline in the current study. As a result of Raman spectroscopy, the differences in peaks represent the protein-related bands in mutant lines having a general decreasing trend in intensity when compared to the commercial cultivar. Amide-I (1,630 and 1,668 cm−1), Histidine, Lysine, Arginine, and Leucine bands (823, 849, 1,241, 1,443, and 1,582 cm−1) showed decreasing wavenumbers. Beta-carotene peaks at 1,153 and 1,519 cm−1 showed increasing trends when the normalized Raman intensities of the mutant lines were compared.