Hemolytic behavior of a series of different categories of Gemini surfactants was determined in their low concentration range. Cationic Gemini surfactants of different molecular architectures prove to be highly cytotoxic even at 0.1 mM. Anionic and amino acid-based Gemini surfactants were minimally cytotoxic, although their toxicity was concentration-dependent. With respect to monomeric surfactants of comparable hydrocarbon chain lengths, cationic Gemini surfactants were much more toxic than anionic Gemini surfactants. Incubation temperature was another important parameter that significantly drove the hemolysis irrespective of the molecular structure of the surfactant. Results indicated that the surface activity or liquid-blood cell membrane adsorption tendency of a surfactant molecule determined the degree of hemolytic anemia. Greater surface activity induced greater cytotoxicity, especially when the surfactant possessed a stronger ability to interact with the membrane proteins through hydrophilic interactions. That provided cationic Gemini surfactants a higher ability for hemolytic anemia because they were able to interact with an electronegative cell membrane with favorable interactions in comparison to anionic or amino acid-based Gemini surfactants. These findings are expected to help in designing surface-active drugs with a suitable molecular architecture that can avoid hemolytic anemia.