Major obstacle in the management of fungal diseases is the availability of rapid diagnostic procedure. The growth-based phenotypic testing is still the method of choice in many microbiology laboratories. More recently, an ion-paired reverse phase chromatography (IP-RP)-based molecular assay has been developed. This molecular approach includes PCR amplification of ribosomal DNA followed by Restriction Fragment Length Polymorphism (RFLP) analysis using the commercially available IP- RP-chromatography system called the WAVE. To make this molecular assay adaptable in a diagnostic laboratory, we developed a computational tool that allows automation of data management and query. This assay is simple, rapid, and has allowed for rapid identification of 27 out of 28 medically important fungi. The use of internal control in the sample and data analysis significantly improved the specificity of this assay. The contribution of this computational approach in reducing the turnaround time for the test is expected to translate into improved patient care.