A two-step protocol to remove endotoxins from recombinant human-like collagen was investigated. To meet the clinical need, Triton X-114 two-phase extraction system was applied as the first step to remove large amounts of endotoxins, while affinity chromatography was used as the second step to achieve further purification. Under the best experimental condition optimized through response surface methodology techniques, endotoxin level was 0.025 similar to 0.25 EU/mg and protein recovery was 81.9%, respectively, which indicated that 99% of endotoxins were eliminated with high protein recovery. Therefore, the two-step protocol can be exploited as an efficient method for endotoxin elimination in biotechnology processes.