Spectrophotometry using a small sample volume has been developed to measure the alkalinity (or bicarbonate). However, the experimental and calculation processes are complicated, and the atmospheric CO2 has to consider in preparing standard solutions. This study aims to quantify the dissolution of the atmospheric CO2 when using spectrophotometry. Also, the effect on the standard solution in titrating that uses to make calibration curve are calculated to understand the effects of the different CO2 concentrations in a laboratory. The difference between the bicarbonate concentrations and the calculated ones based on the updated chemical equilibrium formula was from 0.038 to 5.4×10-6 mg/L. The maximum difference was found at pH 5.0 in the 10 mg/L HCO3- standard solutions. The bicarbonate concentration without the atmospheric CO2 reaction (C1) and with the atmospheric CO2 reaction (C2) was calculated by the PHREEQC. The difference between C1 and C2 ranged from 0.01 to 0.02 mg/L, but the calculated bicarbonate concentrations between the HCl titration and the PHREEQC output were certainly different, which ranged from 3.1 to 11.5 mg/L at the pH 4.3 endpoint. In contrast, at the pH 4.8 endpoint, the difference was significantly decreased from 0.8 to 1.3 mg/L. The effect of the increasing atmospheric CO2 by human breathing in a laboratory is only 0.05 mg/L in the standard solutions when titrating. From the results of this study, the experimental and calculation processes to correct the bicarbonate concentration by the effect of the atmospheric CO2 in a laboratory may be omitted if natural waters are targeted.