This is the first article to report of the chemical profile of the leaves of Rubus alceifolius Poir, a widely distributed medicinal plant in the Eastern Hemisphere. Fresh leaves of R. alceifolius were treated by a set of experimental protocols to prepare a lyophilized aqueous extract. A novel strategy was used to analyze the extract, i.e., The extract was then analyzed using a new strategy, i.e., database-affinity ultra-high-performance liquid chromatography-quadrupole-Exactive-Orbitrap-tandem mass spectrometry (UHPLC-Q-Exactive-Orbitrap-MS/MS). Using MS/MS at full elucidation and comparison with the database, 33 compounds were putatively identified, including 12 flavonoid derivatives, 6 phenolic acid derivatives, 6 caffeoylquinic acids, two tea polyphenols, and others. Especially, 12 isomers have been strictly distinguished, including apigenin vs 2'-hydroxydaidzein, luteolin 7-O-glucuronide vs scutellarin, (+) catechin vs (-) epicatechin, 3-O-caffeoylquinic acid vs 4-O-caffeoylquinic acid vs 5-O-caffeoylquinic acid, and 3,4-O-dicaffeoylquinic acid vs 3,5-O-dicaffeoylquinic acid vs 4,5-O-dicaffeoylquinic acid. In addition, 21 non-isomeric compounds were also found under both negative and positive ion models, such as ellagic acid and gallic acid. Structural comparison suggested that there were biogenetic relationships among the flavonoid derivatives, such as glycosidation and hydroxylation. All these new findings will help to understand the substance basis of the traditional medicinal functions of R. Alceifolius. The structural comparison suggested biogenetic relationships between flavonoid derivatives, such as glycosidation and hydroxylation. These findings will allow the scientific community to better understand R. Alceifolius traditional medicinal functions.