Since a method of rapidly detecting extended-spectrum beta-lactamase (ESBL) production in gram-negative isolates from patients with severe infection is urgently required, the present study of a novel commercial kit was conducted. The Cica-Beta Test I (Kanto Chemical, Tokyo, Japan) is designed for the rapid detection of ESBL in gram-negative bacteria directly from isolated colonies in a 15-min protocol. In this study, a total of 304 strains of Klebsiella spp., Escherichia coli and Proteus mirabilis were tested using the novel kit and the phenotypic confirmatory disk test using cefotaxime and ceftazidime with and without clavulanate. The kit showed 95.5 and 98.1% sensitivity and specificity, respectively, as compared to the disk test, and thus proved to be an appropriate tool for the rapid detection of ESBL.