Our study identified high-molecular-weight compounds from Tityus serrulatus venom (TsV), and most of them have not yet been well explored. TsV was fractionated using FPLC system with different columns, analyzed by SDS-PAGE, and characterized by MALDI-TOF/TOF. Our study showed that TsV contains several high-molecular-weight compounds, including CRISPs, metalloproteinase and hyaluronidase. We show how these molecules can be obtained from TsV, enabling future studies about their molecular structures and biological actions, expanding knowledge about this venom.