Natural products from medicinal plants, either as pure compounds or as standardized extracts, provide unlimited opportunities for new drug leads. Due to an increasing demand for seeking therapeutic drugs from natural products there is a need to develop analytical methodologies for different purposes which include the extraction, isolation and characterization of active ingredients in botanicals and herbal preparations. Various analytical methods available for the isolation, characterization and quantification of active ingredients from plant extracts which include various spectrometric and chromatographic methods along with some hyphenated techniques are discussed. The present review helps to design a method for the isolation, characterization and quantitative determination of major constituents from plants.