Plantago ovata is an important medicinal plant and a rich source of secondary metabolites including polyphenolic compounds. In vitro callus culture of this plant opens up the possibility of photochemical prospecting with enhanced production of some polyphenols using elicitors. Utilizing the callus is advantageous as the whole plant need not be sacrificed. Subculturing results in an increase of callus biomass along with the most prized phytochemicals. The reservoir of polyphenolic compounds has been studied by various authors from time to time to extract polyphenols in general and some others that are unique to Plantago by HPLC analysis, such as trans-cinnamic acid, gallic acid, rutin, quercetin, catechin, luteolin-7-O-β-D glucoside, coumaric acid, vanillic acid, chlorogenic acid, trans-ferulic acid, and caffeic acid. Organic plant additives, such as casein hydrolysate, coconut water, and even plant growth regulators in different concentrations and combinations, have resulted in enhancing polyphenol and flavonoid biosynthesis during in vitro callus culture of this plant. Polyphenols, being natural products, result in higher antioxidant activity in subcultured calli. Epidemiological studies have shown that a human diet rich in polyphenols gives protection against several metabolic disorders, such as diabetes, cancer, and cardiovascular diseases. Polyphenols are intermediate products of the phenylpropanoid pathway and their production is regulated by differential expression of the genes of this pathway, such as phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), and dihydroflavonol-4-reductase (DFR). Another important gene is polyphenol oxidase (PPO), which causes enzymatic browning by oxidation of phenolic compounds to O-quinone and polymerizes polyphenolic compounds. It is regarded as an antioxidative defense enzyme of plants. Critical quantitation of the organic chemicals that enhance the production of polyphenols will be dealt with in this review chapter in relation to PAL, CHS, DFR, and PPO. This will clarify biochemical regulation by using molecular biological techniques like reverse transcription and real-time PCR. Bioinformatic details on the structure prediction of these genes will also throw light on the upregulation of gene expression in relation to increased polyphenol biosynthesis. © 2018 Elsevier B.V.