The use of flavonoids for prevention and cure of human diseases is already widespread. Quercetin shows antiproliferative effects against various cancer cell lines. These aspects made flavonoids an interesting object for industrial production. This paper describes a plant-cell-culture production approach that provides increased yields of flavonoids. Cell culture of Astragalus missouriensis was established, and flavonoid production was determined and optimized. The chemical investigation of cell extracts led to the isolation of different flavonoids by means of HPLC and TLC. The main aglycon identified was Quercetin in both free and bound forms (as glycosides). Isoquercitrine (quercetin-3-0-glucoside) and Quercitrine (quercetin-3-0-rhamnoside) were the main flavonoid glycosides in all tested cell lines. Rutin (quercetin-3-0-rutinoside), Hyperoside (quercetin-3-0-galactoside), Scopoletin and Phenolcarbonic acids - p-coumaric and chlorogenic have been also detected. In order to increase flavonoid production in vitro the effect of plant growth regulators (auxins and cytokinins) and sucrose concentration were examined. Content of flavonoids was severally decreased in higher concentrations of 2,4dichlorophenoxyacetic acid (2,4-D). Addition of cytokinin stimulated both proliferation and flavonoid production. Kinetin was favorable for flavonoid production in A. missouriensis cells. After optimization of production medium maximum total amount of flavonoids 1.34% was achieved. The highest amount of Isoquercitrine (5.3 mg/g DW) and Quercitrine (8.1 mg/g DW) was found in medium with optimal combination of growth regulators naphthaleneacetic acid (NAA) I mg/l, Kinetin 2 mg/l and 6% (w/v) sucrose. Rapidly growing cell lines were selected to increase the efficiency of the production of Quercetin derivatives. These results could provide a practical means of in vitro cultivation of this medicinally important plant and for further biotechnological applications.