Buruli ulcer disease (BUD), caused by Mycobacterium ulcerans, has become the third most common mycobacterial disease worldwide. Antimycobacterial therapy is considered the treatment of choice. With the introduction of antimycobacterial treatment, laboratory confirmation of clinically suspected cases became crucial for the clinical management of BUD. Currently available diagnostic laboratory tests include microscopy, culture, histopathology and IS2404 PCR. Several IS2404 PCR assays were applied for case confirmation in endemic countries, and IS2404 PCR is considered the most sensitive method for the laboratory confirmation of BUD. Due to the extended presence of mycobacterial DNA under antimycobacterial treatment, however, PCR is not suitable for monitoring of treatment success. Currently, cultures are considered the only valid confirmatory test for the detection of viable bacilli.