The present study aimed to improve the processing of data acquired from laser speckle contrast imaging (LSCI) to provide a standardization method to explore changes in regional cerebral blood flow (rCBF) and to determine the correlations among rCBF, cerebral ischemic lesion volume and microvascular density over time in a focal ischemic region. C57BL/6J mice were subjected to focal photothrombotic (PT) ischemia. rCBF was measured using LSCI at different time points before and after PT ischemia through an intact skull. Standardized rCBF (SrCBF), defined as the ratio of rCBF measured in the ipsilateral region of interest (ROI) to that in the corresponding contralateral region, was calculated to evaluate potential changes. In addition, the volume of the ischemic lesion and the microvascular density were determined using Nissl staining and immunofluorescence, respectively. The relationships among the ischemic lesion volume, microvascular density and SrCBF were analyzed over time. The results showed that the cortical rCBF measured using LSCI following PT ischemia in the C57BL/6J mice gradually increased. Changes in the cerebral ischemic lesion volume were negatively correlated with SrCBF in the ischemic region. Changes in the microvascular density were similar to those observed in SrCBF. Our findings indicate that LSCI is a practical technique for observing changes in murine cortical rCBF without skull opening and for analyzing the relationships among the ischemic lesion volume, microvascular density and SrCBF following focal cerebral ischemia. Preliminary results also suggest that the use of LSCI to observe the formation of collateral circulation is feasible.