The spinal cord dorsal horn is a relay hub that receives sensory information from the peripheral nervous system and transmits bioelectrical signals to the brain. In vivo imaging of neuronal and glial activity in the dorsal horn can provide insights into both functions and dysfunctions of the neuronal network in the spinal cord. With multimodal NLO microscopy, we identified a thin-myelin sheath region allowing to image over 200 μm deep below pia with subcellular resolution. By using an optically cleared intervertebral window, dorsal horn neuron and microglia activities can be observed without activating spinal cord inflammation. Two-photon imaging of neurons and microglia as well as the optical clearing improvement at different tissue depths were further characterized over time. Using this inflammation-free imaging method, we conducted a longitudinal study of dorsal horn microglia dynamics following sciatic nerve transection. Furthermore, stable in vivo calcium imaging of neurons in the dorsal horn was performed with electrical stimulation on the mouse’s hind paw. The subcellular-resolution imaging enabled characterization of the distinct calcium transients of neuronal somas and dendrites. This minimally invasive imaging approach to spinal dorsal horn through an optically cleared intervertebral window provides a reliable platform for studying and understanding cellular activities in the spinal dorsal horn.