In 1944, Draize et al., published a paper entitled “Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes”. The Organization for Economic Co-operation and Development published their first guideline on eye irritation in 1981, using rabbits. In the early eighties the development of alternative non-animal tests to replace the Draize eye test started. The first attempts to validate alternative tests for eye irritation were considered to be relatively simple by comparing in vitro and in vivo irritation index scores. In the early nineteen-eighties, we introduced the use of isolated eyes as an alternative test for the Draize eye irritation test. What was expected to be a process of several years, however, turned out to be a decades spanning process still not fully completed. For a large part, this can be attributed to the nature of the in vivo test in rabbits, which is more complicated and compromised than originally believed. This paper describes, most chronologically, the development, performance, validation and application of the Isolated Eye Test and, in broader perspective, the international validation and acceptance of this alternative test by regulatory authorities and agencies.