A simple and direct method without a derivation step for routine analysis of tobramycin has been developed. This method used reversed-phase ion-pair high performance liquid chromatography (HPLC) with a refractive index (RI) detector and a C18 column which is stable at pH above 1.00. The presence of 4.50 mg·mL^-1 trifluoroacetic acid (TFA) in the mobile phase improved the protonation of tobramycin and the formation of ion-pairs, and thus reduced its hydrophility. This unique separation–detection combination showed good linearity with correlation coefficients 0.9996 in the concentration range of 0.25–2.50 mg·mL^-1. The quantitation limit and detection limit were determined to be 0.23 mg·mL^-1 and 0.071 mg·mL^-1, respectively. Tobramycin was recovered in 98.00%, 98.84% and 99.64% for tobramycin solutions at concentrations of 2.25 mg·mL^-1, 1.50 mg·mL^-1 and 0.75 mg·mL^-1, respectively. The relative standard deviations for six spiked samples ranged from 0.20% to 2.40%, indicating a good reproducibility of this method.