Chlamydia abortus is an obligate intracellular, gram-negative bacterium and a significant zoonotic agent associated with abortion storms, particularly in small ruminants. Due to its intracellular lifestyle, conventional isolation is limited, necessitating specialized protocols. This study aimed to develop and evaluate an optimized isolation method for C. abortus by comparing the efficacy of three different cell lines: Vero, monolayer BHK-21 (m-BHK-21), and suspended BHK- 21 (s-BHK-21). Samples were collected from PCR-positive materials, including a calf ?s stomach content, sheep vaginal swab, lamb fetal liver, and calf fetal liver. Each sample was inoculated into the three cell lines. Cell cultures were monitored using an inverted microscope equipped with a trinocular imaging system. Stamp staining and conventional PCR were employed for initial and intermediate detection. Final confirmation of C. abortus presence was performed via Stamp staining and quantitative analysis using Real-Time PCR. Bacterial growth was quantified using a cell counter, leveraging the bacterium?s gram-negative staining properties. Isolation was successful across all cell lines using the calf stomach content sample, whereas the remaining samples yielded positive results in only one cell type. Among the tested lines, s-BHK-21 demonstrated superior performance, producing the highest bacterial load (4.7×10? cells/mL). A 94-fold increase in viable cell count was observed from initial inoculation to the final harvest. The s-BHK-21 cell line is recommended as the most effective platform for C. abortus isolation, providing enhanced bacterial yield and reduced processing time. Its application may improve diagnostic accuracy and facilitate further research into C. abortus pathogenesis and control strategies.