Abstract Introduction: Rhopalurus junceus (R. junceus), recently known as Heteroctenus junceus (H. junceus) scorpion, is one of the endemic species types of scorpion found in Cuba. The scorpion venoms contain a complex of peptides, proteins, and non-protein compounds that make its separation and purification procedures difficult and time-consuming. Applications in basic science, for clinical and biotechnological purposes, lead to characterize the venom of the scorpion employing techniques as electrophoresis gel, chromatographic profile and biological activity, in this case, determining if these results may be influenced by the sex and the captive condition of the scorpion. Materials and Methods: The protein concentration was determined by the modified Lowry method. Differences in protein content were analyzed by the SDS-PAGE technique using Image J 1.46 software. The chromatography profile was obtained by High-Performance Liquid Chromatography (RP-HPLC) employed a C18 reverse-phase column from Shimadzu. HeLa (human cervix adenocarcinoma) and Vero cells (normal African green monkey kidney) are used to determine the cell viability by the MTT assay. Results: No statistically significant differences were observed in the total protein concentrations of the venoms obtained from females and males and the mixture of both sexes. Electrophoretic analysis showed similarities and differences between the experimental groups. The chromatographic profile evidence a similar behavior related to the sex and captivity condition as the scorpion in wildlife. No statistically significant differences were observed between the viable cells of the HeLa and Vero cell lines treated. All the venoms evaluated were capable of inducing morphological changes in the HeLa tumor line.