Mate (Ilex paraguariensis A. St. Hil.) is a raw material used to prepare popular beverages in South America, whose healthy effects have been pointed out. The leaves and stems of mate contain a complex assemblage of saponins, mainly ursolic acid and oleanolic acid glycosides, and their quantification presents difficulties such as weak cromophorous sites and interference of other compounds. On the other hand, the sugar moieties are a chief character that distinguishes them. We developed and validated a high-performance liquid chromatography method using a refractometer detector for quantifying the major saponins in extractive solution of mate leaves. The validation indicates the suitability of the method, presenting linearity in the concentration range between 102.5 to 854.0 microg/mL to matesaponin 1 and between 103.0 to 515.0 microg/mL to matesaponin 3. The repeatability coefficient of variation was 4.70% and 6.61% for matesaponin 1 and matesaponin 3, respectively. The inter-day coefficient of variation was 6.77% and 7.79% for matesaponin 1 and matesaponin 3, respectively. The analysis of plants from Brazilian States Mato Grosso do Sul and Rio Grande do Sul showed the predominance of matesaponins 1, 2, and 3 in the corresponding extractive solution. The sum of these three saponins ranged between 3 and 10 mg/g for mature leaves in the majority of sampled plants.