A simple and sensitive procedure for the determination of gentamicin is presented, based on reaction with o-phthaldialdehyde (OPA) in combination with N-acetylcysteine (NAC) in a basic medium. The use of N-acetylcysteine instead of liquid mercaptans for the formation of isoindole derivatives is a good solution for avoiding the unpleasant odour of thiol compounds. Determination of the resulting fluorescent derivatives was carried out by reversed-phase chromatography at λex=328 nm and λem=423. Stability of OPA/NAC/gentamicin-conjugate was improved by the addition of hydroxypropyl-β-cyclodextrin. The calibration graph was linear (r = 0.9997) over the range 0.4-12.8 μg mL^-1. The precision (RSD%) of the method varied from 1.5 to 8.5. The method was satisfactorily applied to the determination of gentamicin in pharmaceutical preparations with a mean recovery of 100.02% for injections and 88.94% for drops.