Striga hermonthica (Del.) Benth a well-known hemi-parasitic weed, it also has been used widely in African folk medicine to remedy broad spectra of diseases. The current contribution is an attempt to establish reproducible in vitro callusing system. In vitro seedling’s stem segments were used as an explant for callus induction, in 1.5% or 3.0% sucrose added into Murashig and Skoog medium (MS) and supplemented with different auxins, α-Naphthalene-3-acetic acid (NAA), 2,4-dichlorophenoxy acetic acid (2,4-D), Indole-3-acetic acid (IAA), or Indole-3-butryic acid (IBA) at different concentrations each alone or in combination with cytokinin 0.5 mgl-1 6-benzyl aminopurine. The most effective auxin was NAA with maximum 79% callus induction rate. All auxin treatments induced callus in all concentration when used alone or combined with BAP, except 2,4-D, which induced the callus only when combined with BAP. A high sucrose concentration was required for high callus induction rate by all auxin treatments. IAA and IBA auxins induced direct shoot regeneration and had low callus induction rates. NAA, IAA and IBA induced organogenic calli, whereas 2,4-D in combination with BAP induced non-organogenic callus. We further screened preliminarily the phytochemical contents of the callus and intact plant, which was revealed the presence of flavonoids, terpenes, saponins, cardiac glycosides, alkaloids, tannins and coumarins. Experimental data of both seed culture and callus induction could provide a route to further enhance the efficiency of callus initiation of S. hermonthica for medicinal purposes and understanding the infection mechanism of the witch-weed plant.