The callus and hairy root cultures of Prosopis farcta were established to develop effective strategies to enhance its valuable and medicinally important flavonoid compounds. For callus induction, the hypocotyl, cotyledon and shoot explants were subjected to different plant hormones, naphthalene acetic acid (NAA), benzylaminopurine (BAP), kinetin and dichlorophenoxyacetic acid (2,4-D). Greater callus induction was obtained from hypocotyl explants on MS medium containing 3.0 mg L NAA + 2.0 mg L BAP. With the addition of 0.5 mg L asparagine to this medium, the maximum callus growth was achieved. Hairy root culture of P. farcta was performed using transformation of different explants with strains of Agrobacterium rhizogenes LBA9404, A4, AR15834. The AR15834 strain was more effective for hairy root induction where it caused hairy root formation on 59% of the infected cotyledon explants. We compared profiles of flavonoids isolated from seedling roots, hairy roots, and callus cultures of P. farcta. The colorimetric analysis showed that the content of total flavonoids of hairy roots was 1.54 and 2.52 times higher than in seedling roots and callus, respectively. The presence of flavonoids was verified by LC/MS in positive ion mode. The results showed that flavonoid composition was different in the roots and callus. Naringenin was the major constituent in callus, whereas resveratrol, quercetin and myricetin were the most abundant compounds found in hairy roots. The main objective of this research was to establish hairy roots in P. farcta to synthesize flavonoids at levels comparable to in vitro-grown roots. The present study also opens up a way to further improve the production of pharmaceutically valuable flavonoids and to produce desired metabolites using the hairy root culture system.