A precise and sensitive reversed phase high-performance thin-layer chromatography (RP-HPLC) method was developed for the determination of nilotinib (NTB) in spiked plasma, urine, and pharmaceutical capsule formulation. The method was based on derivatization NTB with 4-chloro-7-nitrobenzofurazan (NBD-Cl) in the borax buffer (pH 9). The method employs an isocratic elution using acetonitrile and 10 mM orthophosphoric acid (40:60 v/v) as a mobile phase and an C18 column (4.6 mm × 250 mm, 5 μm, Waters Symmetry), with a fluorescence detector (λex: 447 nm, λem: 530 nm). The method validation was performed with respect to linearity, recovery, accuracy, precision, and stability. The linear ranges were 100–600 ng mL−1 in standard solution, plasma, and urine. Correlation coefficients (r2) were higher than 0.9997 for all of the analytes, indicating good linear relationship. The percentage recovery was 87.89% for plasma, 95.35% for urine, and 96.07% for capsules.