In the present work, High Pressure Carbon Dioxide (HPCD) has been used to inactivate the enzymes Polyphenol Oxidase (PPO) and Pectin Methylesterase (PME). The work has been done in two stages: in the first one, fundamental knowledge about the inactivation mechanism induced by HPCD was obtained using pure commercial enzymes. The influence of the main process parameters was studied: pressure, temperature, time and ratio CO2/volume of enzymatic solution. The changes in the tertiary structure of the enzymes after the HPCD treatment were analyzed by fluorescence spectroscopy running two different tests: intrinsic fluorescence measurement and ANS binding. In the second stage, the acquired knowledge was used to study the inactivation pattern of these two enzymes in liquid and solid natural products, including fruit and vegetable juices or shrimps. The results revealed differences in the inactivation pattern of the enzymes in these substrates compared to the pure commercial enzymes, but the applicability of HPCD technology for the inactivation of enzymes in real substrates at industrial scale was also revealed.