Candidemia caused by Candida albicans and Candida glabrata is constantly increasing, accompanied by a rising use of the few available antifungals. The widespread use of echinocandins and azoles for treatment of invasive candidemia has enhanced the development of antifungal-resistance resulting in an increasing health care problem. Hence, the rapid detection of resistant strains is required. This study aimed to evaluate the detection of C. albicans and C. glabrata strains resistant against caspofungin by MALDI Biotyper antibiotic susceptibility test- rapid assay (MBT ASTRA). This novel semi-quantitative technique facilitates the detection of caspofungin-resistant strains within 6 hours. MBT ASTRA results were compared to the data obtained by the Clinical and Laboratory Standards Institute guidelines (CLSI). Clinical isolates of C. albicans (n= 58) and C. glabrata (n= 57) were analysed by MBT ASTRA and CLSI microdilution. CLSI antifungal susceptibility test against caspofungin classified C. albicans isolates into 36 susceptible and 22 resistant strains, and C. glabrataisolates into 5 susceptible, 33 resistant and 19 intermediate, respectively. For C. albicans, the comparison of MBT ASTRA and the CLSI method revealed an excellent categorical agreement of 100%. Sensitivity and specificity of 94% and 80% between MBT ASTRA and CLSI were detected for C. glabrata strains, based on categorical agreement, respectively. In conclusion, the results obtained by MBT ASTRA indicated that this is a very promising approach for the rapid detection of candida isolates resistant to caspofungin.