Isoflavonoids are interesting class of natural products due to their positive effects on human health. Isoflavonoids include isoflavones, isoflavanones, isoflavans, rotenoids and pterocarpans. Although they are reported from many plant families, most isoflavonoids are produced by the subfamily Papilionaceae of the Fabaceae. Various chromatographic methods have been applied for the purification of isoflavonoids. Simple Ultra Violet (UV) absorption spectra as well as both One and two dimensional NMR (1D-and 2D-NMR) are critical for the identification of isoflavonoids. Each class of isoflavonoids has its unique feature in both 1 H-and 13 C-NMR that enable their proper characterization. High Resolution Mass Spectrometry (HRMS) is a substantial tool in such challenge. In vitro experiments indicated that isoflavonoids possess antioxidant, antimutagenic, antiproliferative as well as cancer preventive effects. Epidemiological studies provide support for some of these effects on human. Members of this class also are reported to have antimicrobial activity. In this chapter, isoflavones, isoflavanones, isoflavans, homoisoflavonoids and isoflavenes will be discussed in relation to their occurrence, methods of purification, spectral characters helpful in structure elucidation as well as their biological importance.