A thorough understanding of the synaptic ultrastructure is necessary to bridge our current knowledge gap about the relationship between neuronal structure and function. Recent development of focused ion beam scanning electron microscopy (FIB/SEM) has made it possible to image neuronal structures with high speed and efficiency. Here, we present our routine protocol for correlative two-photon microscopy and FIB/SEM imaging of glutamatergic synapses. Femtosecond-pulsed near-infrared laser was used to create fiducial marks around the dendrite of interest in aldehyde-fixed tissues. Thereafter, samples were subjected to en bloc staining with rOTO (reduced osmium tetroxide-thiocarbohydrazide-osmium tetroxide), followed by lead aspartate and uranyl acetate to enhance tissue contrast. Reliable detection of postsynaptic density (PSD) and plasma membrane contours by the sample preparation protocol optimized for FIB/SEM allows us to precisely evaluate morphological features that shape glutamatergic synaptic transmission.