Euphorbia peplus, Sisymbrium irio and Malva parviflora are medicinal plants and found in many parts of the world. They have a large number of secondary metabolites that are utilized to treat numerous illnesses. The current study deals with examining the influence of plant growth regulators on callus induction by applying different explants of each of the studied plants. A comparative study was done between the ethanolic extracts of roots, stems and leaves of the mother plants and those regenerated from in vitro to analyze the amount of alkaloids, tannins and flavonoids. The obtained data disclosed that the maximum callus induction %, fresh and dry weights were recorded by root segments of E. peplus on Murashige and Skoog's medium containing 2 mg / l benzyl adenine and 0.5 mg / l 2,4-dichlorophenoxyacetic acid. Stem explants of S. irio achieved the obvious percentage of callus initiation on medium augmented with 0.5 mg / l Kinetin and 2mg/l 2,4-dichlorophenoxyacetic acid. In the case of M. parviflora the best callus initiation as well as fresh dry weight were recorded by root explant on Murashige and Skoog's medium including 2,4-dichlorophenoxyacetic acid alone. The study also showed that ethanolic callus extracts have better synthesis for investigated alkaloids, tannins, and flavonoids. The results demonstrated that different explant types may differ in accordance with species, as a result of different responses of their microenvironments to media ingredients.