To develope insulin MIP and HMR1423 production in yeast Pichia pastoris GS115 as host organism to express insulin. Studying the effects of the optimal temperature and N-sources that produced the highest concentration of both insulin and was detected by Indirect Competitive Enzyme-Linked Immunosorbent Assay (Indirect competitive ELISA). Insulin MIP was expressed the highest concentration at 20℃ and the concentration was 39.74 µg/ml at 84 hr whereas insulin HMR1423 was expressed the highest concentration at 30℃ and the concentration was 32.74 µg/ml at 24 hr. When the optimal N-sources for both insulin production was studied, MIP expressed the highest concentration when yeast extract and peptone was used with the concentration 42.52 µg/ml at 48 hr , while HMR1423 expressed the highest concentration when ammonium sulfate was used with the concentration was 32.74 µg/ml at 24 hr. From this research, the primary structure of proteins are slightly changed in a few amino acids, parameters in protein production in P. pastoris are significantly different. Moreover, the expression of both insulin analogs in this study found in relatively short period of time comparing to the previous studies is a benefit for further development of recombinant insulin production.