Larotrectinib or VITRAKVI is a first-generation pan-tropomyosin-related kinase (TRK) inhibitor. It is a highly selective, tumor-agnostic agent which has been approved in both adult and pediatric patients. Three different analytical methods for estimation of such important drug were developed. Two simple spectrophotometric methods were developed: Method I involves measuring the absorbance of different concentrations of the larotrectinib at 264 nm in concentrations ranging from 2 to 15 μg/mL in ethanol while first derivative peak amplitude (Method II) was derived and measured at 278 nm in concentration ranges from 2 to 15 μg/mL. A spectrofluorimetric technique was carried out by creating an ion pair complex through the interaction of larotrectinib with eosin Y in the presence of a Britton-Robinson buffer at pH 3.5. The fluorescence quenching of the resulting complex was then detected at 535 nm as the emission peak following excitation at 265 nm. The spectrofluorimetric technique exhibited high sensitivity and linearity across concentration ranging between 0.5 and 10 μg/mL. These three methods were effectively utilized to quantify larotrectinib in both its raw form and its pharmaceutical formulation. Greenness of the developed methodologies was concerned and evaluated via three different tools including analytical ecoscale, green analytical procedure index (GAPI), and analytical greenness metric approach (AGREE).