Siraitia grosvenorii (Swingle) C. Jeffrey is widely recognized for its anti-inflammatory properties, as well as its roles in lung purification, phlegm elimination, intestinal function regulation, and anti-tumor activity. Its pharmacological activity is attributed to a diversity of functional components. However, due to the extensive application of sweet glycosides in food additives, there have been few studies on non-sweet glycosides, particularly those with high polarity. This paper investigates the chemical constituents in the non-sweet glycosides fraction of S. grosvenorii juice. First, an MCI GEL CHP20P chromatographic column was utilized to enrich the non-sweet glycosides fraction. Furthermore, two-step medium-pressure liquid chromatography (MPLC) was performed for the efficient preparative separation of high-polarity non-sweet glycosides with similar structures, using C18 and silica gel as stationary phases, respectively. Seven non-sweet glycoside compounds were identified through NMR and mass spectrometry analyses, including three new compounds (4-hydroxyphenylethanol 4-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside, 4-hydroxyphenylethanol 4-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside and n-butanol 1-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside), as well as four known ones (α-D-glucopyranosyl-(1→4)-D-glucose, α-D-glucopyranosyl-(1→2)-β-D-fructofuranoside, methoxy hydroquinone diglucoside, and β-D-glucopyranoside). The results demonstrate that mixed-mode MPLC using different stationary phases is an efficient approach for separating non-sweet glycosides from S. grosvenorii.