Background: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 urine 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 five published studies, Al-Amound et al., (2002) research was the most qualified. The method is validated in the range of 0.5-10 mg / L with a correlation coefficient (r) of 0.998. The detection limit and the limit of quantification of gentamicin in urine are 75 μg / ml and 250 μg / ml, respectively. The coefficient of variation in the precision test was 1.72%, and the percentage of recovery was in the range of 94.3%. Conclusion: This method was repeatable for the analysis of Gentamicin in urine in the concentration of various drugs, making it suitable for monitoring drug therapy and pharmacokinetic studies.