Multicolor fluorescence-activated cell sorting (FACS) has allowed cancer researchers to gain insights into the role of immune cells within the tumor microenvironment. According to this, flow cytometry is widely used to characterize and quantify immune cell infiltration during tumor development in a given tumor implant. In preclinical mouse models of peritoneal carcinomatosis, FACS is usually used to study the peritoneal lavages. However, one of the most relevant tissues to analyze in peritoneal metastases, the omentum, often goes unnoticed. Previous works have demonstrated that flow cytometry analysis of the omentum is critical to understanding the anti-pro-tumoral response of gynecological or gastrointestinal cancer. Here, we set up a detailed protocol to study different cell populations in omental metastasis with specific multicolor FACS panel design, including T cells, natural killer cells, natural killer T cells, and B cells in peritoneal carcinomatosis mice models. In cancer, these cell populations exhibit unique phenotypic profiles and are known to contribute to tumor growth and immune evasion. Nevertheless, immunomodulation of the omentum through intraperitoneal immunotherapy strategies has been demonstrated to be crucial in controlling peritoneal carcinomatosis. The protocol described in this chapter lists the steps to be taken for the localization, extraction, processing, and characterization of different immune cell populations of omental tumor implants in peritoneal carcinomatosis mice models using multicolor flow cytometry. In conclusion, we consider that the immune characterization of omentum cell infiltration is relevant to predicting the antitumor response of different locoregional immunotherapy strategies.