A novel method for the direct determination of the aminoglycoside tobramycin was developed and validated based on reversed-phase high-performance liquid chromatography (RP-HPLC) with evaporative light scattering detector (ELSD). Using a Waters ODS-2 C18 Spherisorb column with an evaporation temperature of 45 degrees C and nitrogen pressure of 3.5 bar, the selected mobile phase consisted of water/acetonitrile 55:45 containing 1.5 mL L(-1) HFBA (11.6 mM) in an isocratic mode at a rate of 1.0 mL min(-1). Tobramycin's retention time was 4.3 min with an asymmetry factor of 1.7. A logarithmic calibration curve was obtained from 1 to 38 microg mL(-1) (r > 0.9998). LOD was 0.3 microg mL(-1); within-day %RSD was 1.0 (n = 3, 4.7 microg mL(-1)) and between-day %RSD was 1.1 (3 days within a week). The developed method was applied to the determination of tobramycin in a pharmaceutical crude substance and formulations (eye drops and ointments). Dilution experiments revealed the absence of interference from excipients (no constant and proportional errors); recovery from spiked samples was 99-103% with %RSD < 2.2 (n = 3x3). The developed HPLC/ELSD method was also found to be applicable in the determination of tobramycin in human plasma (0.6-12.5 microg mL(-1)) and urine (1.5-12.5 microg mL(-1)) after solid-phase extraction using carboxylate cartridges followed by solvent evaporation (x2 preconcentration). A mean recovery of 86% for plasma and 91% for urine was obtained.