Hypoxis (commonly known as African potato, Ilabatheka, Inkomfe, sterretjie or yellow starflowers and also as monna wa maledu or thitidi) is a genus of the family Hypoxidaceae. The rootstocks of Hypoxis species, particularly H. hemerocallidea, are well-known to be used in traditional medicine for the treatment of different ailments, such as urinary tract infection, epilepsy, prostate cancer and onset diabetes. Several visits have been made to the Faraday Medicinal Market and Abey Bailey Nature Reserve in Johannesburg to determine the availability of Hypoxis plant materials. From these visits, it was discovered that different Hypoxis species are harvested and sold as the same plant commonly referred to as the African potato, and the treatment with these plants might be questionable as the secondary metabolites might differ. This was proven when a number of rootstocks bought from the medicinal market grew into plants showing distinct morphological differences when planted at the Agricultural Research Council (Roodeplaat, Pretoria). It is possible that the plants sold are used as the substitutes for the commonly main plant, which is H. hemerocallidea and a reality is that many of the problems related to the quality of medicinal plants are based on the substitution of the declared plants and when the substitution occurs the quality of the plants becomes compromised leading to the risks of public health. It is however, not known whether the substitution is done deliberately or unintentionally since the outward morphology of Hypoxis species are not the same except their bright yellow flowers. The similarity within these species is on their underground rootstock. The dosage and toxicity of plant preparations is extremely important and, therefore adulteration is a concern where plant preparations are taken orally and the information about the plants used not being accurate. The aim of this study was to compare the secondary metabolite content of four Hypoxis species namely, H. acuminata, H. hemerocallidea, H. iridifolia and H. rigidula Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC) were used to analyze the secondary metabolite content of the plant extracts. Differences were also noted as one green compound was observed only in H. acuminata and H. rigidula. The HPLC results showed major differences in retention time in fresh material. The antibacterial activity of extracts of all four Hypoxis species showed similar results, although the activity differed amongst the microorganisms. The species showed high level of antioxidant activity that increased with increasing concentration in all four Hypoxis species. The species also showed no toxicity when tested in vitro on Vero cells however, they seemed to be toxic to cancer cells (Hela cells) but with a higher concentration. Hypoxoside was isolated and identified as the purple colour band on the TLC fingerprint and was confirmed in all the species. It might be possible to replace or substitute different Hypoxis species for H. hemerocallidea for medicinal use as the chromatograms of the other three species correlate well with the chromatograms of H. hemerocallidea after storage.