Mate (Ilex paraguariensis) is an important natural product in the economic and cultural context of Brazil. Peroxidase and polyphenol oxidase have been responsible for quality deterioration and browning in mate. The objective of this work was to investigate a methodology of extraction and enzymatic activity determination of oxidases present in mate tea leaves and to evaluate the oxidases stability. The effects of raw-material mass, buffer molar concentration, Triton X-100 addition, extraction pH, pH activity measurement, polyvinylpyrrolidone K90 addition, and centrifugation time were evaluated by the experimental planning methodology. The storage of the oxidases along 150 days at low temperatures showed that no significant difference was found at -4 and -80 degrees C but significant difference was observed when compared to 4 degrees C. The results showed that higher activities of oxidases are obtained at similar conditions. The exposition to high-temperatures and the variation of the time of exposition affected the enzymatic activity significantly.