The development of pharmaceutical products brought a revolution in human health, but controlling the concentration of drugs in these products is essential to patient’s safety. In this study, a simple, sensitive and specific HPLC method is developed and validated to quantitatively estimate Gilteritinib in pharmaceuticals dosage form. Chromatographic separation was performed on an Hemochrom Intsil C18 Column (250 x 4.6 mm; 5µm) via isocratic elution with mobile phase consisting of 25mm Sodium Perchlorate (pH = 2.0 adjusted with Orthophosphoric acid): acetonitrile (65:35) with a flow rate of 1mL/min. The detection was achieved with UV/Vis detector with a detection wavelength of 310 nm. The method was validated in terms of linearity, sensitivity, precision, accuracy and limit of quantification tests. Gilteritinib can be successfully separated with good linearity (the regression equation is y=41928x + 108090, R2 = 0.9998 and perfect recovery with the results of accuracy were in the range 98-102%.