The importance of adequate remodeling of pre-existing arterial interconnections to form endogenous collateral bypasses—i.e., arteriogenesis—is highlighted in the extensive link between adequate collateral development and enhanced patient outcomes with arterial occlusive disease.1,2 The key initiating stimulus for arteriogenesis is increased shear stress.3 Upon occlusion of a major artery, downstream pressure is reduced, causing an increase in the pressure gradient, blood flow, and shear stress along pre-existing collateral arteries that bypass the occlusion. Both the magnitude4 and duration5 of increased shear stress determine maximal collateral outgrowth and eventual resolution. Nonetheless, direct measurements of shear stress magnitude along individual collateral arteries have not been reported. We believe this paucity of hemodynamic information is due, at least in part, to the lack of a method capable of making such measurements. The primary challenges are the small caliber of the developing collateral vessels (often in an initial ) and the need to integrate these data over a large area. Of the technologies with sufficient spatial resolution, such as multiphoton excitation fluorescence,6,7 optical coherence tomography,8,9 and photoacoustic tomography,10,11 there are still multiple barriers to their adoption for studying arteriogenesis. These include cost and technical expertise, depth of imaging, and data integration over the observed vascular networks. One potential solution is laser speckle flowmetry (LSF) microscopy. Both relative flow and functional microvascular density have been measured by LSF in cerebral,12–15 retinal,13,16,17 and dorsal skinfold window chamber microcirculation. From a theoretical standpoint, absolute LSF measurements are possible,13 and we have recently shown LSF to be an efficient solution for measuring flow changes across large microvascular networks.18 The use of LSF has, however, been limited by the depth of signal acquisition to the most superficial vascular structures.19 In turn, this limits its application for analysis of collateral arteries embedded deeply in the tissue. To increase acquisition depth, a recent report20 suggested that the reorientation of the illumination source to transmission through the tissue could project and capture the hemodynamic signals from deeper vascular structures. Here, we developed a trans-illumination-based LSF system to measure, for the first time, the in vivo spatial distribution of collateral artery hemodynamics before and after femoral artery ligation (FAL) in the mouse ischemic hindlimb, which is the most widely used model of peripheral arteriogenesis.