Daptomycin is known as the first lipopeptide antibiotic shows activity against gram-positive bacteria. A fast, simple and repeatabile RP-LC method has been developed for the determination of daptomycin in mobile phase solution and human urine. This drug was exposed to various stress conditions such as hydrolytic, oxidative, thermaland photolytic and the stressed samples were detected by the proposed method. Daptomycin was well separated on a X-Terra RP-18 column using the mobile phase consisting of a mixture of acetonitrile:water (45:55 v/v) at a flow rate of 1 mL min-1. The system operates at 222 nm for daptomycin and 319 nm and tinidazole was used as internal standard under these conditions. The linearity of daptomycin was obtained between 0.50 and 12 µg mL-1 concentration range. Limits of detection was determined as 0.1176 and 0.1412 µg mL-1 and the limits of quantification was obtained as 0.3563 and 0.4277 µg mL-1 in the mobile phase and urine, respectively. Hydrochloric acid, sodium hydroxide, hydrogenperoxide affected response of daptomycin under stress degradation conditions.