Anastomotic leakage (AL) is a serious complication occurring after esophagectomy. The current knowledge suggests that inadequate intraoperative perfusion in the anastomotic site contributes to an increase in the AL rate. Presently, clinical estimation undertaken by surgeons is not accurate and new technology is necessary to improve the intraoperative assessment of tissue oxygenation. In the present study, we demonstrate the application of a novel optical technology, namely Single Snapshot imaging of Optical Properties (SSOP), used to quantify StO% in an open surgery experimental gastric conduit (GC) model. After the creation of a gastric conduit, local StO% was measured with a preclinical SSOP system for 60 min in the antrum (ROI-A), corpus (ROI-C), and fundus (ROI-F). The removed region (ROI-R) acted as ischemic control. ROI-R had statistically significant lower StO% when compared to all other ROIs at T15, T30, T45, and T60 ( < 0.0001). Local capillary lactates (LCLs) and StO% correlation was statistically significant (R = -0.8439, 95% CI -0.9367 to -0.6407, < 0.0001). Finally, SSOP could discriminate resected from perfused regions and ROI-A from ROI-F (the future anastomotic site). In conclusion, SSOP could well be a suitable technology to assess intraoperative perfusion of GC, providing consistent StO% quantification and ROIs discrimination.