A highly sensitive high-performance liquid chromatography method has been developed using the pre-column fluorescent derivatization of daptomycin (DAP) through cyclization of the amino group of ornithine with 2,3-naphthalenedialdehyde. With the proposed method, the limits of detection and quantification of DAP in murine serum were 8 and 3 nmol/L, respectively, and the calibration curve was linear across the examined dynamic range from 8 nmol/L to 1 μmol/L (n = 8, r = 0.9986). This method is suitable for animal experiments examining the side effects of DAP therapy using mice as a simple method with quantification to the order of 10 nmol/L.