Ciguatoxins (CTXs) are highly toxic and poorly available low molecular weight hydroxylated polyether compounds synthesized by the benthic dinoflagellate Gambierdiscus toxicus Adachi and Fukuyo. They are transferred via the food marine web from the benthos to herbivorous and carnivorous fish and causes after ingestion of contaminated fish a human syndrome called ciguatera. For both immunization and assay purposes, it is necessary to covalently couple low molecular weight compounds (i.e. haptens) to immunogenic macromolecules, however difficulties arise from the lipophilic nature of CTXs and the absence of a functional group on these molecules. Using a brevetoxin congener (PbTx-3) as a model for hydroxylated polyether compounds, this work describes a new methodology for immunogen preparation dealing first with the micro-scale preparation of a toxin hemisuccinate derivative (PbTx-3 HS) and its subsequent conjugation to carrier proteins in a reversed micellar medium, with emphasis on the elucidation of optimal conditions for both synthetic steps. Second, considering previous demonstrations that the epitope density of the conjugates, the immunization schedule and the antibody repertoire can greatly influence the induction of specific antibodies (amount, class and affinity), two mice and a rabbit were immunized. The specificity and the affinity of antibodies raised against PbTx-3-BSA conjugates are presented and discussed. These results confirmed 1) the potential in preparing and characterizing immunogen with only 0.5 µmol of toxic compounds 2) the immunogenic properties of the conjugate 3) the applicability of this entire procedure to generate antibodies to ciguatoxins.