Background: To date, no standardized protocols nor a quantitative assessment of the near-infrared fluorescence angiography with indocyanine green (NIR-ICG) are available. The aim of this study was to evaluate the timing of fluorescence as a reproducible parameter and its efficacy in predicting anastomotic leakage (AL) in colorectal surgery. Methods: A consecutive cohort of 108 patients undergoing minimally invasive elective procedures for colorectal cancer was prospectively enrolled. The difference between macro and microperfusion (ΔT) was obtained by calculating the timing of fluorescence at the level of iliac artery division and colonic wall, respectively. Results: Subjects with a ΔT ≥ 15.5± 0.5 s had a higher tendency to develop an AL (p < 0.01). The ΔT/heart rate interaction was found to predict AL with an odds ratio of 1.02 (p < 0.01); a cut-off threshold of 832 was identified (sensitivity 0.86, specificity 0.77). Perfusion parameters were also associated with a faster bowel motility resumption and a reduced length of hospital stay. Conclusions: The analysis of the timing of fluorescence provides a quantitative, easy evaluation of tissue perfusion. A ΔT/HR interaction ≥832 may be used as a real-time parameter to guide surgical decision making in colorectal surgery.