To establish a universal analytical tool that could be used as a bioactive quality determination procedure on medicinal plant extracts, a range of spectrophotometric assays, HPLC, near infrared reflectance spectroscopy and chemometric analysis were employed for determination of the bioactive quality of 16 widely spread medicinal plants. Macro-constituents (total carbohydrates, soluble polysaccharides, proteins, amino acids) and secondary plant metabolites (total phenols, flavonoids, hydroxycinnamic acids, flavons, and flavonols) were determined, and HPLC method for the simultaneous determination of phenolic acids and flavonoids was developed, and its linearity, limits of detection and quantification, precision, and accuracy were validated. The evaluated medicinal plant extracts were characterised by a marked protein (marigold-4.22 g/kg dw), amino acid (marigold-61.14 g/kg dw), and carbohydrate content (dandelion-113.5 g/kg dw), while Lamiaceae plants were distinguished as the predominant sources of polyphenolic bioactives (<2.26 g GAE/L). The developed HPLC method enabled separation of 24 polyphenolic compounds within a short analysis time (30 min) and revealed rosmarinic and chicoric acids as the prevalent polyphenolic constituents. NIR spectroscopy coupled with chemometric analysis of all determined analytical parameters indicated the suitability of NIR analysis for amino acids, carbohydrates, and polyphenols determination in medicinal plant extracts.