Soluble microneedles (MNs) have recently become an efficient and minimally invasive tool in transdermal drug delivery because of their excellent biocompatibility and rapid dissolution. However, direct monitoring of structural and functional changes of MNs in vivo to estimate the efficiency of insulin delivery is difficult. We monitored the dissolution of MNs to obtain structural imaging of MNs' changes by using optical coherence tomography (OCT). We also observed the effect of MNs on microvascular conditions with laser speckle contrast imaging (LSCI) and measured the blood perfusion of skin to obtain functional imaging of MNs. We determined the performance of two soluble MN arrays made from polyvinyl alcohol (PVA) and polyvinyl alcohol/ polyvinylpyrolidone (PVA/PVP) by calculating the cross-sectional areas of the microchannels in mouse skin as a function of time. Moreover, the change in blood glucose before and after using MNs loaded with insulin was evaluated as an auxiliary means to demonstrate the ability of the soluble MNs to deliver insulin. Results showed that the structural imaging of these MNs could be observed in vivo via OCT in real time and the functional imaging of MNs could be showed using LSCI. OCT and LSCI are potential tools in monitoring MNs structural and functional changes.