Several liquid chromatographic (LC) methods have been described for the analysis of gentamicin. LC combined with pulsed electrochemical detection (LC-PED) or evaporative light scattering detection (LC-ELSD) was found to be the most suitable technique. A first method, previously developed by Adams et al. used a poly(styrene-divinyl benzene) stationary phase with a mobile phase containing sodium sulphate, sodium-1-octanesulphonate, tetrahydrofuran, 0.2M phosphate buffer (pH 3) and water. However, the polymer columns show low efficiency, which also leads to poor sensitivity. So, recently the use of newer conventional C18 columns was further investigated. Improved separation was obtained using a Supelcosil LC-18-DB column with an adapted mobile phase. Another method derived from a company method was checked by using a Gemini column and a mobile phase containing an aqueous solution of trifluoroacetic acid (TFA) and pentafluoropropionic acid (PFPA) adjusted to pH 2.6 with sodium hydroxide (NaOH). This method was transferred to ELSD by replacing the non-volatile NaOH with volatile ammonium hydroxide solution. A volatile method, which was originally developed for ELSD using an aqueous solution of 50mM TFA and gradient elution with methanol, was also combined with PED. In this study, these methods were compared with regard to their selectivity, sensitivity and ease of use.