Twenty-one species belonging to 13 genera of Acanthaceae obtained from Saudi Arabia were studied using electrophoretic analysis for protein patterns and two isoenzymes polymorphism in addition the phytochemical screening by TLC and HPLC. A total number of twenty-three protein bands observed between the species ranged 10 and 250 K Da, nine esterase and six peroxidase polymorphisms were detected. The biochemical data (protein profiles and isoenzymes polymorphisms) considered a significant tools for the relationships between the studied species. Furthermore, the phytochemical screening including the total contents of alkaloids, flavonoids, terpenoids and saponins were demonstrated the presence of alkaloids, flavonoids in all studied taxa with a variable contents and absence of total terpenoides and saponins in some species. As well, fourteen phenolic acids have been screened by high-performance liquid chromatography (HPLC) method. Gallic acid, phenol, protocatechuic acid, p-coumaric acid and o-coumaric acid in substantial amounts. Both the biochemical and the phytochemical data were conducted by means of the numerical analyses based on in total 38 electrophoretic characters and 18 phytochemical contents. On the basis of UPGMA clustering analysis, the two recognition distinct taxonomic groups and several clusters were distinguished. The existent results are useful for evaluating the relationships between the studied Acanthaceae species both at subfamilies and tribe levels.