A rapid HPLC-UV method was developed for the quantitative analysis of kojic acid contained in fungal agar media. The methanol extract from fungal agar media was diluted with acetic acid aqueous solution and injected into HPLC. The separation was achieved on a C18 column within 10 minutes in an isocratic mode. The mobile phase was composed of water, methanol, and acetic acid. Spike and recovery tests were done using three representative agar media, and accuracy, precision and limit of detection were preferable. The developed method was applicable for the analysis of kojic acid in two Aspergillus strains practically, showing the usability of this method to evaluate the fungal ability to produce a beneficial secondary metabolite, kojic acid.