Background: Peritoneal surface malignancies (PSM) include peritoneal metastases from cancers such as colorectal and ovarian cancer, as well as mesothelioma, which is treated with cytoreduction surgery (CRS) and its completeness is a critical determinant of survival. This feasibility study evaluates pegulicianine, a fluorescent imaging agent, in combination with two fluorescence imaging systems to detect PSM. One system, previously shown to detect residual cancer in the lumpectomy cavity following removal of the main specimen, is referred to as pegulicianine fluorescence-guided surgery (pFGS). The second is a commercially available laparoscope, referred to as the RED System modified to support both pegulicianine fluorescence and white-light (WL) imaging. This study aims to determine the optimal dose and timing of pegulicianine administration, and correlate imaging findings between pFGS and the RED System. Here, we report imaging results from patients who underwent intraoperative imaging with pFGS and the RED System. Trial Design and Methods: Patients undergoing abdominal CRS were eligible for enrollment. Pegulicianine was administered intravenously prior to surgery at doses of 1.0 mg/kg (n = 3), 1.5 mg/kg (n = 9) or 2.0 mg/kg (n=6). During surgery, the surgeon identified regions of interest (ROI) within the peritoneum comprising suspected metastatic lesions and adjacent normal tissue. Each ROI was imaged in situ using pFGS, and some with the RED System, at 60±30-minute intervals or until resection. Imaging findings were correlated with histopathology, and tumor-to-normal fluorescence signal ratios (T:N) were calculated. The effect of administration time was evaluated by plotting T:N for each organ as a function of time from pegulicianine administration to image acquisition. Results: Six patients were dosed at the pre-specified levels and imaged with pFGS alone for initial feasibility. Interim analysis demonstrated mean (± SD) T:N of 1.65 (± 0.11) and 1.87 (± 0.09) for the 1.0 mg/kg (n=3) and initial 1.5 mg/kg (n=3) cohorts, respectively, supporting the feasibility of in vivo cancer detection using pFGS. Based on these results, the study was expanded to enroll 6 additional patients at a dose each of 1.5 mg/kg and 2.0 mg/kg. All patients were imaged with pFGS. Four patients dosed at 2.0 mg/kg were imaged with the RED System. Image panels were generated for each tissue and time point, including WL and intraoperative fluorescence images, and red-channel fluorescence images with applied tumor and normal tissue masks for quantitative analysis. RED System images demonstrated mean (± SD) T:N of 1.71 (± 0.32) across organs imaged 2-6 hours after pegulicianine administration, with T:N increasing over time. Conclusion: These results support further evaluation of pegulicianine with laparoscopic imaging using the RED System for solid tumor detection.