Novel and accurate analytical methods were developed and validated for the characterization of Fluoxetine in its pure and pharmaceutical dosage form Prozac®. Fluoxetine was determined by IBA techniques (PIGE, PIXE and RBS). It has been also analyzed spectrophotometrically at 610 nm after oxidation with potassium permanganate in alkaline medium. In addition, Fluoxetine was kinetically determined using the initial rate method, the fixed absorbance method and the fixed time method. Moreover, a Gas chromatography - mass spectrometry technique is proposed for the investigation of Fluoxetine without a prederivatization phase. The spectrophotometric method was performed with a concentration array of 2-10 μg/mL at 610 nm and a regression coefficient (r) of 0.996. The fixed time method was the most suitable one to determine Fluoxetine with correlation coefficient value (r) of 0.9966. The Gas chromatography - mass spectrometry investigated the drug in a concentration range of 20-100 μg/mL and a regression coefficient (r) of 0.999. IBA analysis presented a precision of less than 3% and a very low limit of detection. Consequently, these proposed methods would be useful tools for determining Fluoxetine as all the assay results exposed satisfactory sensitivity, accuracy and reproducibility