To demonstrate the importance of calibration measurements in 3 Tesla proton magnetic resonance (MR) spectroscopy (1 H-MRS) thermometry for human brain temperature estimation for routine clinical applications. In vitro proton MR spectroscopy to obtain calibration constants of the water-chemical shift was conducted at 3 Tesla with a temperature-controlled phantom, containing a pH-buffered aqueous solution of N-acetyl aspartate (NAA), creatine (Cr), methylene protons of Cr (Cr2), dimethyl silapentane sulfonic acid (DSS), and sodium formate (NaFor). Estimations of absolute human brain temperature were performed utilizing the correlation of temperature to the water-chemical shift for the resonances of NAA, Cr, and Cr2. Data for calibration of the metabolites' chemical shift differences and in vivo temperature estimations were acquired with single-voxel point-resolved spectroscopy (PRESS) sequences (repetition time/echo time = 2000/30 ms; voxel size 2 • 2 • 2 cm 3). Spectroscopy data were quantified in the time-domain, and a Pearson correlation analysis was performed to estimate the correlation between the chemical shift of metabolites and measured temperatures. The correlation coefficients (r) of our calibration measurements were NAA 0.9975 (-0.0609), Cr-0.9979 (-0.0621), Cr2-0.9973 (-0.0577), DSS-0.9976 (-0.0615), and NaFor-0.8132 (-2.348). The mean calculated brain temperature was 37.78-1.447°C, and the mean tympanic temperature was 36.83-0.2456°C. Calculated temperatures derived from Cr and Cr2 provided significant (p = 0.0241 and p = 0.0210, respectively) correlations with measured temperatures (r = 0.4108 and r =-0.4194, respectively). Calibration measurements are vital for 1 H-MRS thermometry. Small numeric differences in measured signal and data preprocessing without any calibration measurements reduce accuracy of temperature calculations, which indicates that calculated temperatures should be interpreted with caution. Application of this method for clinical purposes warrants further investigation and a more practical approach.