Cholangiocarcinoma is a lethal tumour of the bile ducts. Cholangiocarcinoma fibroblast growth factor receptor gene fusions forms can be targeted by Pemigatinib (PGT). PGT a recently approved kinase inhibitor by the US-FDA, has its approval accelerated due to the disease viciousness. Development of a sensitive yet available and economical analytical platform to quantify PGT in human plasma is genuinely needed. Enabling monitoring of the therapeutic plan, hence, ensuring the drug efficacy and safety through pharmacokinetic studies. High-performance liquid chromatography with fluorescence detector (HPLC-FD) method is proposed using the native fluorescence of PGT. PGT and seliciclib (internal standard) chromatographic conditions optimisation, revealed favourable use of isocratic mobile phase consisting of methanol:ammonium acetate buffer (70:30v/v, pH5.0) pumped into C18-column (150 mm length × 4.6 mm internal diameter, 5 μm particle size), at 1 mL min