The current gold standard for the laboratory diagnosis of Mycobacterium ulcerans disease (Buruli ulcer) is a PCR-based assay. In the endemic African countries this assay is established only at a few central reference centers. Therefore, there is urgent need for the development of a rapid diagnostic test. The macrolide toxin mycolactone produced by M. ulcerans represents an ideal target for such a test, as it is specific for this pathogen. By using a protein conjugate of a truncated non-toxic synthetic mycolactone derivative, we succeeded to generate for the first time mycolactone-specific monoclonal antibodies as a prerequisite for the development of a mycolactone-specific competition ELISA. In this assay, a selected antibody is bound to a solid support as capturing antibody. If a clinical specimen contains mycolactone, it is competing with a mycolactone-derived reporter molecule for binding to this antibody, allowing to detect the toxin at low nanomolar concentrations. In collaboration with Drugs & Diagnostics for Tropical Diseases (DDTD), reagents and parameters generated and optimized for the competition assay were used to convert the ELISA format into a lateral flow assay intended for use at peripheral health stations. Together with the Foundation for Innovative New Diagnostics (FIND), DDTD and the Nagasaki University we received a grant from the Global Health Innovative Technology Fund (GHIT) for the field validation of a first mycolactone-specific prototype rapid diagnostic test for Buruli ulcer.