For many fungal infections,
in vitro
susceptibility testing is used to predict if an isolate is resistant or susceptible to the antifungal agent used to treat the fungal infection. For
Madurella mycetomatis
, the main causative agent of mycetoma,
in vitro
susceptibility testing currently is not performed on a routine basis. The current
in vitro
susceptibility testing method is labor intensive and sonication must be done to generate a hyphal inoculum. For endpoint visualization, expensive viability dyes are needed. Here we investigated if the currently used
in vitro
susceptibility method could be adapted to make it amendable for use in a routine setting which can be used in low income countries, where mycetoma is endemic. First, we developed a methodology in which hyphal fragments can be generated without the need for sonication, by comparing different bead beating methodologies. Next,
in vitro
susceptibility was assessed using standard broth microdilution assays as well as disc diffusion, E-testing and VIPcheck™ methodologies. We demonstrate that after a hyphal suspension is generated by glass bead beating, disc diffusion, E-testing and VIPcheck™ can be used to determine susceptibility towards itraconazole, posaconazole and voriconazole of
Madurella mycetomatis
. The MICs found with the E-test were comparable to those obtained with our modified CLSI-based broth microdilution
in vitro
susceptibility assay for itraconazole and posaconazole. Furthermore, we found an inverse relationship between the zone of inhibition and MIC obtained with E-test and the modified CLSI broth microdilution technique.