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Background Rutin is a natural flavonol that showed excellent antiglycation activity with an IC50 value of 294.5 +/- 1.5 mu M. In the current study, three selected plant species of Euphorbia, i.e., Euphorbia helioscopia, Euphorbia larica, and Euphorbia wallichii, were analyzed for the quantification of rutin. Methods The quantification was done through a newly developed method of Emission spectroscopy coupled with Partial Least Square Regression (PLSR) and UV-visible spectroscopy as a parallel cross-validation method. Results The spectroscopic results indicated the highest rutin concentration in the roots of E. helioscopia (11.25 mg/100 g) followed by roots of E. wallichii (9.93 mg/100 g), leaves of E. helioscopia and the whole plant of E. larica (9.41 mg/100 g). The leaves of E. wallichii (8.66 mg/100 g) were found to contain the lowest concentration of rutin among all the tested samples. Conclusion The present method is one of the simple, robust, and non-destructive methods to carry out the quantitative estimation of rutin in plants.