Gymnema sylvestre is an important medicinal plant, but a method for preparing standardized extract enriched with biomarkers is not available. The practiced crude hydro-alcoholic extracts of G. sylvestre contained a few percentage of gymnemic acids (GAs). The enrichment of GAs in preprared extracts is essential from a pharmacology point of view. Presently, a scale-up process has been developed for preparing G. sylvestre standardized extract (Gym-EEXT). Crude extract (Gym-Crude) was prepared in 10-kg batches to obtain a yield of 268.1 mg/g, but GAs was merely 9 mg/g (GA-IV 6.7 mg/g, and GAXVII 2.3 mg/g) in 30 % ethanol–water solvent at 50 ℃ in 5 h. Further, Gym-Crude was processed through polymer-matrix-adsorption techniques for getting Gym-EEXT (150.9 mg/g) with improved percentage of GAs (>10 %) comprised of deacylgymnemic acid (DGA: 8.9 mg/g), gymnemagenin (2.3 mg/g), GA-IV (78.6 mg/g), and GA-XVII (10.2 mg/g). For safety aspects, the pesticide residues, solvent residues, heavy metal, aflatoxins, and mutagenicity in Gym-EEXT were below the detection limits. Hence, Gym-EEXT was enriched with GAs, and fully safe with compliance according to USP-561. An insignificant effect of Gym-EEXT on vitals of experimental mice was observed in acute and sub-acute oral-toxicity experiments. Gym-EEXT significantly improved the blood glucose metabolism, and hence, it potentially reduces hyperglycaemia. Unique chromatographic protocols were developed for the isolation of biomarkers on a 1-kg scale from Gym-Crude with yield of gymnemagenin (5.5 %), and DGA (4.6 %). Finally, a novel, scientifically validated, and the scale-up process has been developed for the preparation of Gym-EEXT along with an exclusive protocol for isolating its biomarkers.