Here, we describe microplate assays for determining the specific activities of four enzymes that constitute the ascorbate-glutathione cycle: APX, MDHAR, DHAR, and GR. In plants, these enzymes play a major role in detoxifying reactive oxygen species produced in cells under environmental stress. This work presents the development of plate reader assays to allow rapid analysis of the ascorbate-glutathione cycle activity using tomato fruits subjected to salt stress as a model. With this method, it is possible to analyze easily in one day the activities of the four enzymes for 30 experimental samples, all in triplicate and with blanks.