Background: Many studies have shown the benefits deriving from plants secondary metabolites to human health also for the prevention of several diseases associated to oxidative stress including cancer, cardiovascular diseases and diabetes mellitus. Nowadays there is a high attention on plant secondary metabolites and analytical techniques related to their identification and characterization. Conclusion: This review aims to report analytical procedures, instrumental configurations, and extraction approaches devoted to secondary metabolites analyses. In this direction, an introduction to secondary metabolites is made with emphasis on their formation, biological and pharmaceutical roles. Extraction, characterization and analytical procedures are described in the second part of this review, while a comparison of the analytical techniques will be discussed in the third part. This review could provide a better understanding of the analytical procedure and techniques for secondary metabolites and their applications in the scientific and industrial areas including food, food supplements, cosmetic, and pharmaceutical, especially related to quality control analyses and to chemical fingerprint elucidation, all in order to standardize these plant-derived extracts before in vitro and in vivo studies.