Momordica balsamina (M. balsamina) is a medicinal herb comprising health-promoting secondary metabolites. This study was aimed to profile bioactive compounds in the methanolic extract of M. balsamina leaves using molecular spectroscopic (UV-Vis and FTIR) and hyphenated chromatographic (UHPLC-qTOF-MS) techniques and evaluate the biological (in vitro anti-inflammatory and cytotoxicity) activities of the extract. The preliminary phytochemical screening tests revealed the presence of cardiac glycosides, flavonoids, saponins, tannins, and terpenoids. The UV-Vis profile revealed various absorption bands ranging from 200 to 750 nm, indicating the presence of flavonoids, phenolic compounds, tannins, terpenoids, carotenoids, chlorophyll, and alkaloids. FTIR spectra confirmed the presence of alkaloids, flavonoids, terpenes, anthraquinones, and phenolic compounds. A high-resolution and accurate mass spectrometer (LC-QTOF-MS model LC-MS-9030 instrument) was used, and the results confirmed the presence of flavonoid aglycones, such as quercetin, isorhamnetin, and kaempferol, as well as pseudolaroside A and dicaffeoylquinic and feruloyl isocitric acids. To the best of our knowledge, this is the first report of pseudolaroside A dimer and feruloyl isocitric acid in M. balsamina leaves. In vitro cytotoxicity assay showed that the extract was nontoxic against human colorectal adenocarcinoma (HT29 and Caco2), Vero, and RAW 264.7 cells. However, the extract showed anti-inflammatory activity on RAW 264.7 cells. The study confirmed that M. balsamina leaves contain nontoxic secondary metabolites that may play a pivotal role in human health as anti-inflammatory agents.