Amphibians at early stages of their life cycle constitute a very valuable ecotoxicological tool for toxicity studies as indicators of environmental quality for wildlife protection purposes. AMPHITOX was developed in 1999 in order to cope with the increasing diversity of chemical and physicochemical agents and cover the needs for a sensitive, cost-effective and customized assay for different exposure times and developmental stage toxicity tests. Based on a set of bioassays using amphibian embryos, a large number of toxicity studies with both environmental samples and physicochemical agents (single or in combination) were included. More recently, the growing concern about the chronic effects of xenobiotics in environmentally relevant concentrations led us to expand the exposure conditions of classic, acute (up to 96 h of exposure), short-term chronic (168 h) and chronic (14 days) AMPHITOX tests with young larvae, to evaluate noxious effects on specific stages of the early life cycle (pulse-exposures) and exposure periods that cover whole stages of development (embryo–larval and metamorphosis). This last test has even included post-exposure effects to assess alterations on metamorphosis, gonadogenesis and sexual differentiation. In this contribution, the basic conditions of AMPHITOX test and some case studies are included to illustrate both basic and customized potential of this toxicity test. The versatility of this test has been challenged with different conditions, customizing it to the toxicity of the sample, exposure conditions, and developmental stage.