Background: Cassia auriculata (Avaram) is an important medicinal plant in India Improved awareness in medicinal flowers has led to an increased need for efficient extraction methods and screening of flavonoid derivatives. Objective: To standardization of extraction solvent system and Characterization of flavonoids through positive and negative electrospray ionization mode using LC-MS/MS from Cassia auriculata extract. Materials and Methods: The different solvents like Methanol, Water, Acetonitrile, Ethyl Acetate, Ethanol, Chloroform, Hexane, Acetone Diethyl ether used frot he identification of flavonoids (Gallic acid, Theanine, Theobromine, Theophylline, Caffeic acid, Caffeine, Ferulic acid, Theacrine, Catechin, Quercetin, EpiGallo Catachin, catechin gallate, Epicatachin gallate and Quercetin hexoside. Results: Based on the peak area percentage the extraction solvent was standardized. The percentage of relative & absolute intensity of screened flavonoids was observed using LC-MS in positive and negative electrospray ionization. The results show that the methanol extract has more percentage of peak area, relative intensity and absolute intensity. The MS results showed that the negative ionoization has more intensity values of flavonoids and the signal-to-noise ratio was high in negative ionization mode compare to positive mode. Conclusion: Based on the results the methanol is the suitable extraction solvetnt and negative ionization mode of ESI-LC-MS/MS was appropriate for the screening of flavonoids on Cassia auriculata flower extracts.