BACKGROUND: Photo-thermal therapy (PTT) has been at the center of attention as a new method for cancer treatment in recent years. It is important to predict the response to treatment in the PTT procedure. Using magnetic resonance spectroscopy (MRS) can be considered a novel technique in evaluating changes in metabolites resulted from PTT. METHODS: In the present project, we conducted an in vivo study to assess the efficacy of H-MRS as a noninvasive technique to evaluate the response to treatment in the early hours following PTT. The BALB/c mice subcutaneously bearing tumor cells (CT26 cell line) were scanned by H-MRS before and after PTT. Iron oxide-gold core-shell (FeO@Au) as PTT agent was injected into intra-peritoneal at first and then irradiated by NIR laser. Single-voxel Point RESolved Spectroscopy (PRESS) sequence (TE = 144) was used, and metabolites alternations were evaluated by the non-parametric Wilcoxon test. Besides, Nanoparticle (NP) relaxometry was conducted for negative contrast agents' potentials. RESULTS: MRS choline (Cho) peak dramatically reduced 24 h post-PTT (p = 0.01) and lipid peak as a marker for necrosis of tumor elevated (p = 0.01) just in group 3 (NPs injection + laser irradiation) 24 h after the procedure. CONCLUSION: H-MRS showed its potential as a method in detecting the changes in metabolites and revealing the outcome accurately. Response to photo-thermal therapy evaluation was achievable only one day after PTT and proved by a 10-day follow-up of the tumor size. Iron oxide-gold core-shell can also be used as a negative contrast agent in MRI images during therapy.