Abstract: The symbiotic association between corals and zooxanthellae forms the nutritional and structural foundation of coral reef ecosystems. Accurate determination of zooxanthellae density is critical for studying this symbiotic relationship. However, conventional processing methods for algal liquid often face challenges such as incomplete separation of algal cells and algal cell aggregation, significantly compromising measurement accuracy, as zooxanthellae live in the endoderm cells of polyps. In this study, three chemical reagents-TritonX-100 (TRI), ampicillin (AMP), and sodium dodecyl sulfate (SDS) were applied in pairwise combinations alongside physical oscillation to obtain algal suspension from Duncanopsammia peltata. By comparing the differences in zooxanthellae density following the treatments, we investigated the effects of different processing methods on measurement accuracy of zooxanthellae and identified the optimal protocol. Results demonstrated that both chemical and physical treatments significantly increased measured zooxanthellae densities. Among the chemical treatments, SDS treatment yielded the highest zooxanthellae density while markedly reducing algal cell concentrations in supernatants and minimizing algal clumping in the final algal liquid and physical oscillation significantly reduced both the number and area of algal clumps, effectively alleviating algal cell aggregation. The combination of SDS treatment and physical oscillation achieved the highest zooxanthellae density and was identified as the optimal processing protocol. Thus, it enhances the precison of zooxanthellae density for coral physiological and ecological studies.