The Deriphat 2-DE was used to visualize polyphenol oxidase (PPO) isoforms of Moscato and Prosecco grape extracts, partially purified and characterized. Catecholase has similar values in the two varieties, whereas Moscato cresolase data are almost 54% higher. In the first dimension, the PPO of both varieties may be detected by SDS-PAGE, but native PAGE (N-PAGE) gave negative results. For this reason, the samples were solubilized in the zwitteronic detergent Deriphat, which was also included in the gel and the cathodic buffer. Deriphat migrated together with the cathodic buffer, maintaining protein solubility and revealing the PPO profiles of Moscato and Prosecco extracts in native conditions. The combination of Deriphat-PAGE (D-PAGE) and SDS-PAGE (2-DE) also resulted in improved separation efficiency in resolving PPO and specialized stains in evaluating PPO activities. The control, represented by IEF for the first-dimensional separation, had a lower number of spots, demonstrating the higher capacity of Deriphat 2-DE to isolate PPO isoforms from grape extracts. The Deriphat 2-DE method described here is simple but powerful, and the resulting information will be a useful tool for further proteomic research.