<p>Obtaining in vitro plant cultures free of parasites or pathogens, namely axenic cultures is a major challenge both in the field of scientific research and in agronomy or biotechnology. For aquatic macrophytes, plants free from contamination are required for aquaculture or aquariums, and necessary to prevent microbial growth in medium containing dissolved organic carbon. Then, various protocols have been elaborated to facilitate the production of different qualities of plant cultures from “clean” to sterile. The performance of various calcium hypochlorite axenisation protocols in aquatic plants was evaluated. Through trials with variations in hypochlorite concentration and exposure time, it became possible to draw up a response table for three first species (<i>Ceratophyllum demersum</i>, <i>Lemna minor</i> and <i>Myriophyllum spicatum</i>). Additional results on other species were used to define the response of different species to axenisation, so that each species could be oriented towards a particular technique. A trial with successive axenisation steps was thus successfully tested on the two branchy but resilient species (<i>C. demersum</i> and <i>M. spicatum</i>). The effectiveness of protocol variations (e.g. addition of sucrose and use of rinsing breaks during hypochlorite exposures) were also evaluated. This resulted in recommendations that we propose, primarily the banning of sugars during the early stages of axenisation, contrary to previous recommendations in literature protocols.</p>