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.