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The objective of this study undertaken was to acquire maximum information by the shake flask studies for Ascomycin production with Streptomyces hygroscopicus subsp. ascomyceticus ATCC 14891. The morphological observation of culture on plate media was carried out visually and under stereo microscope. The colonies obtained were granular and attained grayish black color on maturity. The microscopic examination of mycelia was performed at 20× and 100× (oil immersion) magnification. To improve the Ascomycin production in shake flask studies, two different approaches were evaluated. In the first set of experiments, “Two stage Fermentation” approach was adopted wherein; matured lab inoculum was transferred directly in different production media. The second approach consisted of set of experiments with “Three stage Fermentation” which involved transfer of grown lab inoculum into seed media and grown seed culture into production media. The age of seed culture was investigated for the betterment of the yield. The maximum concentration of ascomycin obtained was 0.5 mg/gm in the second approach on selected P8 media. Media selection was done on the basis of carbon source, nitrogen source, trace salts, buffer to maintain pH throughout the process and precursor as supplement. Simultaneously, microscopic observation was captured in the present study. Thus it could be concluded that “Three stage Fermentation” on selected media was the best option for scale up of ascomycin.