Microtiter assay is a widely used method for drug susceptibility evaluation of pathogenic fungi in broth and provides a standardized, reproducible and efficient approach for fungal susceptibility testing. This method is important for the determination of the minimum inhibitory concentration (MIC) of antifungal drugs and helps in the development of treatment strategies against fungal infections. The assay is based on exposing fungal cells to different concentrations of antifungal agents in a liquid medium and monitoring their growth (Berkow et al, Clin Microbiol Rev 2020). In a typical microtiter assay, fungal spores or cells are inoculated into wells of a microtiter plate containing serial dilutions of the antifungal agent in a suitable growth medium, such as RPMI-1640. The plates are incubated at specific optimal conditions, usually between 25 and 35 °C, for 24–72 h, depending on the fungal species. Growth is assessed either visually by observing turbidity or quantitatively by spectrophotometric measurements at specific wavelengths. The MIC is determined as the lowest concentration of the antifungal agent that inhibits visible fungal growth or reduces metabolic activity (EUCAST, Clin Microbiol Infect 14(4):398–405, 2008). The scalability of the microtiter test, the minimal reagent requirement and the adaptability to high-throughput formats make it the preferred choice for susceptibility testing of clinically important fungi such as Candida sp. and Aspergillus sp. The ability to check eight drugs/agents in separate rows/series, the scalability of the microtiter test, the minimal reagent requirement and the adaptability to high-throughput formats make it the preferred choice for susceptibility testing of clinically important fungi (CLSI, Reference method for broth dilution antifungal susceptibility testing of yeasts; approved standard-third edition, CLSI Document M27-A3. Clinical and Laboratory Standards Institute, Wayne, 2008). Despite its advantages, the test also has its limitations. These include potential variability in inoculum preparation, drug solubility issues, and the challenge of testing slow-growing or biofilm-forming fungi. Nevertheless, the microtiter assay remains an indispensable tool for the evaluation of antifungal drugs and contributes to the understanding of fungal resistance mechanisms and the optimization of therapeutic regimens.