The antitrypanosomally active crude extract of the sponge Hyrtios sp. was subjected to metabolomic analysis using liquid chromatography coupled with high resolution electrospray ionization mass spectrometry (LC-HR-ESIMS) for dereplication purposes. As a result, a new alkaloid, hyrtiodoline A (1), along with other four known compounds (2-5) were reported. The structures of compounds 1-5 were determined by spectroscopic analyses, including 1D and 2D nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectrometry (HRESI-MS) experiments, as well as comparison to the literature. We further investigated the antitrypanosomal activity of the five compounds, where compound 1 exhibited the most potent antitrypanosomal activity, with a half-maximal inhibitory concentration (IC50) value of 7.48 µM after 72 h.