In this article, a fast, simple, accurate and selective HPLC method was validated for the qualitative and quantitative analysis of the main flavonoids (rutin, hesperidin, catechin, epicatechin, naringenin, quercetin, and kaempferol) in Mexican lime and was appliedto monitor the variation of them during progression of witches’ broom disease of lime (WBDL).The separation of flavonoids was achieved on a C18 column (25 mm×4.6 mm I.D., 5 μm), with a gradient elution composed of two solvents,at a flow rate of 0.7 mL/min and280 nm as wavelength. The method was evaluated with respect to ICH guidelines and then the flavonoids were quantified.The validated method was selective, so that flavonoids were well separated from each other with good resolution. This method showed an excellent linearity (r2> 0.99), high accuracy (recovery), with other validation data, including precision, specificity, limit of detection (LOD), limit of quantitation (LOQ), and robustness. The analysis of flavonoids during progression of WBDL showed that after 30 days of inoculation, levels of flavonoids were considerably increased, but significantly decreased after 120 days. These changes in the levels of metabolites are in good agreement with the activity of phenyl alanine ammonia-lyase (PAL). According to the results, the validated method highly is valuable for quantification analysis of flavonoids during the progression of WBDL and can be effective for diagnosis of healthy plant from infected plant.