Objective: A study aimed at the validation of a high performance liquid chromatography (HPLC) method for detection and quantification of ivermectin (IVM) in commercial milk. Method: After a liquid-liquid extraction with acetonitrile and hexane, a derivatization with a mixture of trifluoroacetic acid and trifluoroacetic anhydride was performed. Chromatography conditions were a C8 column, a fluorescent detector (Ex. 360; Em. 470 nm), a run time of 25 minutes and an isocratic elution at 30°C. The methodology was validated in terms of selectivity, linearity, precision, recovery, limit of quantification (LoQ), limit of detection (LoD), and robustness. Results: The method showed an adequate linearity and selectivity (r 2 =0.999) with an elution time of 13.909 minutes. However, the repeatability and intermediate precision showed RSD values above of 20% and recovery between 37 to 79%. The calculated LoD and LoQ were 2.50 and 5.00 ng/ml respectively. Robustness showed a significant variation on the analytical method with small changes in heating time during the derivatization and flow rate of chromatography system. Conclusion: The HPLC-fluorescence method showed results partially satisfactory and could be the used for IVM detection in commercial milk samples.