Objective To establish a method for determining the recovery of rocuronium microdialysis probe by LC-MS/MS, so as to investigate the stability and reproducibility of microdialysis probe recovery during in vivo and in vitro microdialysis trials and to provide evidence for in vivo microdialysis. Methods The concentration of rocuronium in dialysate was determined by LC-MS/MS and the probe recovery was calculated.The effects of different drug concentrations (50, 200, and 500 ng/mL) and flow rates(0.5, 1.0, 2.0, 3.0, and 4.0 μL/min)on the probe recovery were determined by incremental (dialysis) and reduction (retrodialysis) methods. The in vivo probe recovery in SD rats was determined by reduction method, and its result was compared with that of the in vitro trial. Results The in vitro probe recovery decreased with the increase of flow rate within a range of 0.5-4 μL/min under the same condition. At the same flow rate, different concentrations of rocuronium had little influence on the probe recovery. Under the same condition, the in vitro recovery obtained by incremental and reduction methods were approximately equal and showed a good stability. The in vivo probe recovery obtained by reduction method was similar to the in vitro one. Conclusion Microdialysis can be used for pharmacokinetic study of rocuronium, and retrodialysis method can be used to study probe recovery of rocuronium in vivo.