The enhanced production of prodigiosin was performed by two steps approaches. Here in this study, optimization of preservation condition and methodology for seed culturing were applied to attain a higher yield of prodigiosin using Serratiamarcescens ATCC 14756 in a batch process. Results showed that the yield of prodigiosin was increased while the strain was kept refrigeration for 1 day before subculturing. It was evident that the higher yield of prodigiosin was found with a single time subculturing of previously 24 h preserved strain inside the refrigerator. The maximum yield of prodigiosin is found when the pH of seed media is kept at 6 and the pH of the seed medium was finally shifted after 24 h of subculturing to 7.5. The above final pH (7.5) was considered as a marker for the completion of the seed culturing process. It was evident from results that the production of prodigiosin was found maximum when seed culture was kept inside the refrigerator for 24 h for shocking. The results showed that 1.68 fold higher production of prodigiosin was found by two steps strategies. It was found that enhance the production of prodigiosin was possible by two steps strategies using Serratia marcescens ATCC 14756. Therefore, the above strain could be exploited commercially for the production of prodigiosin.