With the changing epidemiology and emergence of antifungal resistance among Candida species, rapid antifungal susceptibility testing (AFST) is crucial for optimization of antifungal therapy. This study was conducted to standardize a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI -TOF MS) based AFST method (ms-AFST) for susceptibility of Candida tropicalis isolates. Clinical isolates of C. tropicalis were confirmed for fluconazole resistance by the CLSI (M27-A3) method. The incubation period and drug concentration were optimized to determine the minimal profile change concentration (MPCC) by MALDI-TOF MS. The data were analyzed first by direct visual observation of the spectra followed by composite correlation index (CCI) matrix analysis, virtual gel analysis, and cluster analysis for confirmation. Finally, the correlation between minimum inhibitory concentrations (MICs) and MPCCs was evaluated. A total of 15 fluconazole resistant (MICs ranging from 16 to 128 μg/ml) and 19 fluconazole susceptible C. tropicalis isolates (MIC ≤1 μg/ml) were included in this study. All C. tropicalis isolates had significant spectral changes after 4h incubation with fluconazole. Of 34 isolates, MPCCs and MICs were equivalent for 16 isolates, and the MPCC was one dilution lower than the respective MIC in the remaining 18 isolates. This finding was further supported by visual analysis, CCI matrix analysis, virtual gel and principal component analysis dendrogram analysis. The correlation between MPCC and MIC was significant (P < .05). Therefore, a MALDI-TOF MS based AFST assay may be used as a rapid screening technique for fluconazole resistance in C. tropicalis.