Studies on the CO2 system in alpine lakes are performed by scientists of different areas and, therefore, it is crucial to stablish common procedures to investigate the chemical properties of these ecosystems in order to reach comparable results and perform meaningful long-term studies. In this work, a robust procedure was developed to determine the total alkalinity in high altitude mountain lakes which allows the determination of the CO2 system parameters with improved precision and accuracy. In the potentiometric titration of the samples with HCl, used for the Total Alkalinity determination, the fitting between experimental and calculated titration points was greatly improved by incorporating the contribution of new acid-base species which had not been accounted for in previous works. This methodology can be used not only for alpine lakes but also for other natural water systems where the contribution of an extended set of acid-base species is normally not considered. A modified script has been also developed for the Matlab version of the CO2SYS program which allows an adequate calculation of the rest of the CO2 system parameters. The calculated pKa values of the new acid-base species are consistent in nearly all the lakes, ranging from 6.5 to 7, suggesting the similar nature of the acid-base species in all the lakes and that in the Ayous area a different number and/or type of species could be present. Furthermore, the values of all the chemical parameters studied were explained considering the geochemistry of the bedrock.