Two prototype eco-friendly 96-microwell spectrophotometric methods with high throughput for the determination of pemigatinib (anticancer drug) in Pemazyre^® tablets were developed. The two methods involved in microwell one-step formation of colored charge transfer complexes upon interaction of pemigatinib, as an electron donor, with two different benzoquinone electron acceptors: 2,5-dihydroxy-3,6-dichloro-1,4-benzoquinone (chloranilic acid) and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone. The molar ratios of charge transfer complexes, determined by Job’s method, were found to be 1:1. Computational density functional theory analysis of charge transfer complexes confirmed a π→π* single-electron transfer mechanism. An additional hydrogen bond in the chloranilic acid complex was established, further stabilizing the assembly. Conversely, in the 2,3-dichloro-5,6-dicyano-1,4-benzoquinone complex, only the π-stacking interaction is present, with the intramolecular NH···N hydrogen bond remaining intact. The interactions were performed on 96-well transparent plates, and the absorbances of the charge transfer complexes were measured at 530 and 465 nm for pemigatinib–chloranilic acid and pemigatinib–2,3-dichloro-5,6-dicyano-1,4-benzoquinone complexes, respectively. The microwell spectrophotometric method procedures were refined and validated following the International Council of Harmonisation (ICH) standards. The limits of detection of the methods were 8.5 and 4.1 µg well⁻¹ for chloranilic acid and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone methods, respectively. These methods proved highly reliable for assessing pemigatinib in Pemazyre^® tablets and ensuring uniformity. The eco-friendliness/greenness of the microwell spectrophotometric methods was confirmed by three distinct metrics. The methods’ one-step reactions and capacity for processing numerous samples simultaneously contribute to their high-throughput capabilities. In conclusion, this study represents the first two facile, green, and high-throughput microwell spectrophotometric methods for pemigatinib analysis for quality control purposes. In addition, it is the first work exploring the interactions of pemigatinib with chloranilic acid and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone.