In plasmid DNA (pDNA) production from Gram-negative bacteria, endotoxin has been known as the major contaminant. The separation becomes difficult due to its ability to form a stable complex with pDNA apart from sharing common properties like surface charge, molecular size, temperature and pH stability. This study focused on the analysis of zeta potential of endotoxin, theoretical number of cation bound per molecule of endotoxin as well as binding tendency of cations towards endotoxin in the presence of pDNA. These analyses were conducted under various experimental conditions such as types of divalent metal cation, cation concentration, pH and incubation time. The analysis of zeta potential at different cation concentrations and pH values showed that Mg²⁺ had the most significant effect on endotoxin surface charge. The zeta potential of endotoxin reduced by a magnitude of 43.55 mV, from -43.53 mV to 0.02 mV in the presence of 2.0 M Mg²⁺, and a magnitude of 44.12 mV , from -43.53 mV to 0.59 mV at the lowest pH level. However, in the analysis of theoretical number of cation bound per molecule of endotoxin, Zn²⁺ showed the highest number (0.6) compared to Ca²⁺ (0.12) and Mg²⁺ (0.05). The tendency of Zn²⁺ to preferentially bind with endotoxins forming larger aggregated structure was also evident in the DNA gel electrophoresis and transmission electron microscopic analysis.