The development of cancer mouse models is still needed for the identification and preclinical validation of novel therapeutic targets in colorectal cancer, which is the third leading cause of cancer-related deaths in Europe. The purpose of this study was to determine the most accurate tumour cell injection method to obtain suitable peritoneal metastasis (PM) for subsequent therapeutic treatments. Here, we grafted murine colon carcinoma CT-26 cells expressing luciferase into immunocompetent BALB-c mice by intravenous injection (IV group), subcutaneous injection (SC group), intraperitoneal injection after peritoneal scratching (A group) or intraperitoneal injection alone (IP group). Tumour growth was monitored by bioluminescence during the first 15 days post-grafting. The peritoneal carcinomatosis index was evaluated macroscopically, histology, immunohistochemistry and multiphoton microscopy were performed in peritoneal tumour tissue. Upon implantation, no tumour growth was observed in the IV group, similar to the non-injected group. Both the IP and SC groups showed intermediate growth rates, but the SC group produced only a single subcutaneous nodule. The A group exhibited the highest tumour growth at 15 days post-surgery. Anatomic and histologic analyses corroborated the existence of various tumour nodules, and multiphoton microscopy was used to evaluate tumour fibrosis-infiltrating cells in a non-pathologic peritoneum. In conclusion, limited PM was obtained by IP injection, whereas IP injection after peritoneal scratching led to an extensive PM murine model for evaluating new therapeutics.