The purpose of this study was to investigate and compare the cytotoxicity of glyphosate alone or included in Roundup 3 plus modulated by the cytoprotective effects of additional antioxidants such as Vitamin C and Vitamin E on the human keratinocytes cell line HaCaT. An experimental design which allows to minimize the number of experiments was carried out to determine the optimal conditions for cytoprotection against herbicide-induced toxicity. It was shown that HaCaT cell line provides a useful model to study components with toxicity or antioxidant activity. Our results indicated that (i) glyphosate-based formulations can be responsible for oxidative damage to human epidermal cells, (ii) antioxidant compounds should be associated to herbicide formulations to decrease their deleterious effects on human skin. The use of an experimental design connected with the simplex method can be consider to be a fast technique to classify, with a limited number of experiments, the respective role of five parameters in the in vitro cytoprotection by antioxidants of herbicide-induced toxicity.