Objective: The objective of this study was to find the best base mixture composition (poloxamer 407 and HPMC) of chloramphenicol in situ gel formula based on in vitro property (Cumulative amount of drug release). Methods: The in vitro diffusion of chloramphenicol in situ gel study was carried out using franz diffusion cells to know the effect of the Critical Process Parameters (CPPs) as independent variables (poloxamer 407 and hydroxypropyl methylcellulose (HPMC)) on the Critical Quality Attribute (CQA) as dependent variable (cumulative amount of drug release) with 22 factorial design. Results: 22 factorial design of chloramphenicol in situ gel yielded 4 variations of poloxamer 407 and HPMC bases component in %w/v as follows, F1 (5:0.45), F2 (10:0.45) F3 (5:1) and F4 (10:1). The amount of drug release results from in vitro dissolution assay were 30.60% (F1), 45.64% (F2), 58.30% (F3), and 22.50%) (F4). Conclusion: Formula 3 (F3) was considered as the best formula component in terms of in vitro assay of chloramphenicol in situ gel with a desirability value of 0.58.