Abstract: Streptomyces sp. B10 is the isolate of soil samples in the agri-business area of Krian, Sidoarjo, which has great potential as antibacterial. Butanol extract of fermented broth of Streptomyces sp. B10 indicates an antibacterial activity. In the previous study, metabolite separation of the butanol-methanol extract of Streptomyces sp. B10 metabolite by Thin Layer Chromatography (TLC) method has not been satisfied yet.This study aims to establish an appropriate mobile phase to properly separate the compounds in the butanolmethanol extract of Streptomyces sp. B10 metabolite in the elution process using HighPerformance Thin Layer Chromatography (HPTLC) or Reversed-phase Thin Layer Chromatography (TLC). Furthermore, this study also aims to discover the number of compounds in the butanol-methanol extract of Streptomyces sp. B10 metabolite which can be separated by HPTLC or reversed-phase TLC. Lastly, it is to determine the relative concentration of each compound in the butanol-methanol extract of Streptomyces sp. B10 metabolite is based on the comparison of measurement results area with a densitometer.The type of research to be conducted was experimental research. This study separated the compounds in butanol-methanol extract of Streptomyces sp. B10 metabolites. It would be optimized with TLC using RP-TLC, HPTLC, and the eluent optimization to get better results and determine the compound proportion contained in the extract with densitometry.HighPerformance Thin Layer Chromatography of 0.1 M KOH impregnation with a mobile phase of methanol: water (6:4 v/v) could be used for the separation of the butanol-methanol extract of Streptomyces sp. B10 metabolite. It is necessary to research with other methods or instruments to determine the structural composition of compounds in the extracts and determine the types of antimicrobial compounds produced by Streptomyces sp. metabolite.