The quality of botanical food is increasingly assessed by the content of multiple bioactive compounds. This study reports, for the first time, an HPLC fingerprinting method for the quality evaluation of Rubus suavissimus leaves possessing multiple bioactivities. Five constituents, gallic acid, rutin, ellagic acid, rubusoside, and steviol monoside, were quantified and used in developing qualitative chromatographic fingerprints. The limits of detection and quantification ranged from 0.29 to 37.86 mug/mL. The relative standard deviations (RSDs) of intra- and interday precisions were no more than 3.14 and 3.01%, respectively. The average recoveries were between 93.1 and 97.5%. The developed method was validated in the analysis 14 leaf samples with satisfactory results. The contents of the five marker compounds accounted for an average of about 6% w/w with a variability of 16% among the 14 samples collected from a single site and year. Gallic acid was the least, whereas steviol monoside the most, variable compound among the 14 leaf samples. The characteristic compound rubusoside that is responsible for the sweet taste accounted for 5% of leaf weight. The validated method can now be used to quantitatively and qualitatively assess the quality of R. suavissimus leaves as traditional beverage or potential medicines.