Gracilaria is a genus of red-algae found mainly in Asia. The population of Gracilaria spread throughout Indonesia’s marine waters, especially in coastal areas. To date, the unique protein expressions using proteomic approach moreover in Central Java area, Indonesia is sporadic. Nonetheless, there has been no report mentioned about Gracilaria spp. amino acid sequences nor its characteristics. Based on the background mentioned, we aimed to conduct protein characterization by using peptide sequence analysis LC-HRMS. Liquid chromatography coupled with high-resolution mass spectrometry (LC–HRMS) use has growing across biomedical and environmental sciences, hence provides high accuracy for the detection. We followed the LC-HRMS standard protocol, with optimized precipitation standard. TCA/Aceton precipitation of protein were used as protein purification, then the precipitate subjected for Trypsin digestion. Result of protein analysis were described as protein concentrations, molecular model, and peptide sequences. This experiment showed four identified sequences derived from Rhodophyta database, there are TKKILDK (845.5455 Da), TVKSLLTK (889.5717 Da), ILVKTLK (814.5761 Da), TGcGRSKR (921.4683 Da). This study uncovers peptide sequences for Gracilaria spp. and its similarity with other red algae species along with the function of whole peptide sequences. Furthermore, amino acid models of secondary structure Hidden Markov Model(HMM) were provided to support our findings.