Abstract Green assessment of analytical procedures has become an environmental obligation in equivalence to their International Council of Harmonization analytical validation obligation. Worldwide awareness of our planet and ecological hazards have raised the shades of green and sustainable chemistry in pure or formulated API assays. The Green Analytical Procedure Index (GAPI) is instant five pentagrams for evaluating the greenness of each step in the developed analytical procedure, in discriminative colors: green, yellow, and red. In the present study, GAPI is applied to assess three novel direct analytical methods: spectrophotometric, fluorimetric, and high-performance thin-layer chromatography (HPTLC) for remdesivir (RDV) quantitation, both in bulk and pharmaceutical vials. Furthermore, a comparative green level calculated GAPI study has been assembled for the proposed methods versus the previously reported methods, for RDV assay, of similar techniques. Spectrophotometric direct Amax method at 240 nm, fluorimetric emission at 404 nm upon excitation at 275 nm as well as the HPTLC densitometric measurement using ethanol and distilled water (7:3, v/v) as mobile phase, all three methodologies are developed, optimized, and fully validated for RDV quantitation. They have been applied to assay RDV pharmaceutical vials and results are compared together with a one-way ANOVA test. Satisfactory recoveries and nano-level sensitivities in addition to the least standard deviations encourage the use of developed methods for routine analysis in quality control laboratories. Their promising greenness profile satisfies the beliefs of ecological sustainability of Green Agenda 2030.

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