The aim of this study was to evaluate the effect of the depth of correlation (DOC) on the cross-correlation method (CC) applied to microcirculatory blood flow in vitro. The crosscorrelation algorithm was optimized to compute red blood cell velocity profiles in tube flow. Flow rates, estimated by computing the circular integral of mean velocity profiles, were compared with calibrated pump flows for different focus planes of the microscope and different flow rates. Results show a mean flow underestimation of 2.8 ± 5% for all positions of the focus plane inside the tube diameter, highlighting the non-negligible DOC effect when CC is applied to study blood microcirculation. An underestimation of 1% was found with an optimal focus plane. To conclude, flow rate estimation in microcirculatory blood flow can be accurate if the DOC effect is properly compensated for by the CC flow estimation method. Index Terms — red blood cells, out-of-focus microscopy, intravital microscopy, blood flow estimation, microvessels.