Stevia rebaudiana, a perennial herb in the family Asteraceae, is a popular natural sweetener with sensory and functional attributes that outperform those of conventional high-potency sweeteners. Stevia rebaudiana leaves contain sweet glycosides that have biological effects. The various glycosides included in extracts must be measured since, in addition to the major glycosides, they may also contribute to activity. The isocratic HPLC method was validated for the identification of dulcoside A, steviolbioside, rebaudioside C, and rebaudioside B. The development and validation of the HPTLC technique for the simultaneous measurement of three steviol glycosides in Stevia rebaudiana leaves-steviolbioside, stevioside, and rebaudioside-A-also employed the ICH (International Conferences on Harmonisation) guidelines. Near-infrared reflectance spectroscopy (NIRS) was used to assess the steviol glycosides (SGs) content (w/w) dry basis in Stevia rebaudiana leaves using high-pressure liquid chromatography (HPLC) as the reference technique. The noncariogenic and caloric sweeteners known as steviol-glycosides, which are present in the leaves of Stevia rebaudiana, may have had positive effects on human health. Despite the fact that steviol-glycosides are generally regarded to be safe, investigations on animals have shown that the aglycone steviol can have negative consequences. 2D NMR methods and ultra-high performance liquid chromatography with electrospray ionisation mass spectrometry (UHPLC-MS) were used to discover steviol glycosides. The steviol glycosides were then quantified by NMR using anthracene as an internal standard.