Hemocytes are a key player since they actively participate, either through direct or indirect involvement, in the immunological responses of invertebrates. Numerous studies have been conducted on classifying shrimp hemocytes into different subpopulations. Moreover, several methods have been used to understand the hemocytes in crustaceans: ultrastructural microscopic observation, percoll gradient centrifugation, and single-cell RNA sequencing. As a result of this inconsistent classification of crustaceans, it still needs to be further investigated because this means that the hemocytes of crustaceans have yet to be well classified. Therefore, in this study, we separate the hemocyte subpopulations using flow cytometry cell sorting to understand the immune activity of white shrimp (Penaeus vannamei) hemocytes. Flow cytometric analysis (Fig. 1) showed that the presence of four hemocyte subpopulations of white shrimp, differing not only in size (FSC) but also in the degree of granulation (SSC) categorized as the hemocyte 1 (H1), hemocyte 2 (H2), hemocyte 3 (H3), and hemocyte 4 (H4). The FSC and the diameter size showed that there were 4 different sizes of cells that are 9.25 µl (H1), 10 µl (H2), 11 µl (H3), and 12.5 µl (H4). Based on the SSC, two major groups of hemocyte subpopulations could be distinguished: the agranulocyte group, referred to as H1 and H2, and the granulocyte group, referred to as H3 and H4. Moreover, at the transcriptional level, immune-related genes have different functions in each hemocyte subpopulation, such as LGBP, CTL-2, ProPO1, ProPO2, Serine protease, SOD, GPx, CP, Tgase, etc. Further research is needed to explore the immune reaction of each hemocyte subpopulation after injection with a pathogen to better understand the immune response of hemocytes.