Valeriana officinalis L is the most cost-effective plant in Europe. Due to its cardiovascular, anxiolytic, sedative, and anticonvulsant properties, the root of Valeriana is extensively employed germinated in vitro by soaking for 24 hours in 150 ppm Gibberellic acid (GA3) further, cultivated in half MS (Murashige and Skoog salts medium with vitamins) medium. Treatment of MS media with 5 ppm of Benzyl a Dichlorophenoxy acetic acid (2,4-D) increased fresh and dry weights of calli produced from leaf explants. However, supplemented MS media with 3 ppm BA and 1 ppm 2,4 augmentation of MS-medium with 0.1 ppm Indole was accomplished by using peat moss:vermiculite (1:1). Valerenic acid and valtrate were analyzed qualitatively and quantitatively by HPLC in mother plant, callus, regenerated shoots, roots, and Dormival medication which used as a control. The findings showed that regenerated shoots had the greatest concentration of valerenic acid across various extracts (3.80±0.82mg/g DW), whereas in vitro-derived roots from leaf explants contained the highest concentration of valtrate (3.82±0.23mg/g DW).