The prevalidation strategy was applied to evaluate UV-vis spectrophotometric procedure with Folin-Ciocalteu's reagent for polyphenols determination. Favourable prevalidation characteristics verified this procedure as a valuable tool in polyphenols analysis and it was successfully applied for determination of total polyphenols and tannins in leaves, stems and flowers of Plantago L. species growing in Croatia. The results showed the variety of total polyphenols content between different plant parts (leaves: up to 10.15%; stems: up to 4.34% and flowers: up to 5.56%). The content of tannins in stems was from 0.28% to 1.00%, while leaves and flowers contained tannins in concentrations of 2.26% and 2.21%, respectively. The results of polyphenols determination were evaluated by using multivariate analysis (UPGMA and PCA) as a contribution to elucidation of relations between different taxa of genus Plantago L.