INTRODUCTION: Spinal motoneurons represent the final common pathway of the motor system. They are not static elements of the network, but alterations in the levels of physical activity or pathological mechanisms are known to affect their electrophysiological properties. Sharp intracellular recordings are the firmly established gold standard for studying motoneuron excitation/inhibition patterns in vivo in adult animals; however, until recently this technique was not available for mice. We’ve set out to create an electrophysiological setup that would allow us to perform stable intracellular recordings of mouse motoneurons in vivo in order to take advantage of numerous genetic models available for this organism. METHOD(S): Animals were anesthetized with a mix of fentanyl/medetomidine/midazolam, and a complex surgical procedure was performed, that included catheterization of the jugular vein, insertion of a tracheal tube, exposure of the triceps surae (TS) nerve, and Th13‑L2 laminectomy. Sharp glass microelectrodes were inserted into the spinal cord using motorized micromanipulator, and TS nerve was stimulated with constant current pulses in order to evoke motoneuron antidromic activation. Upon successful penetration, TS motoneurons were identified based on an “all or nothing” appearance of action potential, and passive, threshold and synaptic properties were recorded using intracellular stimulation/amplifier. At the end of the experiment, mice were euthanized with overdose of barbiturates. RESULTS: Our approach enabled us to record 2-5 motoneurons in a single experiment with stability ensuring precise measurement of passive membrane properties, intrinsic excitability, and synaptic excitation. CONCLUSIONS: We prove the feasibility of performing stable intracellular recordings of mouse spinal motoneurons in vivo, which should pave the way for future studies of motoneuron plasticity under physiological or pathological conditions. FINANCIAL SUPPORT: This work was supported by NCN grant no. 2017/26/D/NZ7/00728.