Gentamycin is an aminoglycoside antibiotic that is widely used for the treatment of infections, but has an ototoxic and nephrotoxic adverse effect especially in patients with kidney disorders. The mechanism of toxicity is unknown, therefore monitoring of drug concentrations in plasma is needed to enable the correct dosage adjustments to achieve optimal therapeutic levels compatible with safety. Method: In vitro a method for determining Gentamicin levels using High Performance Liquid Chromatography (HPLC) and derivatization processes has been developed. Results: From the six published studies, (Stead & Richards, 1996) research was the most qualified. The method is validated in the range of 0.2-5 mg / L with a correlation coefficient (r) of Gentamicin C1 r2 = 1,0000; Gentamicin C1a r2 = 1,0000; Gentamicin C2 r2 = 0,9999; Gentamicin C2a r2 = 0,9999. The detection limit and the limit of quantification of gentamicin are 10 μg / L and 50 μg / L and the percentage of recovery of Gentamicin C1 96,8%; Gentamicin C1a 99,0%; Gentamicin C2 97,8%; Gentamicin C2a 93,9%.. Conclusion: This method was repeatable for the analysis of Gentamicin in plasma in the concentration of various drugs, making it suitable for monitoring drug therapy and pharmacokinetic studies.