The paper reports the evaluation of the feasibility of using internal standard method for the    determination of nicotine recovery in microdialysis in vitro.  This in vitro experiment included two conditions.  Nicotine and codeine phosphate were dissolved in Ringer’s solution.  Nicotine, codeine phosphate and the   mixture of them were perfused through the CMA30 linear probe separately to calculate the proportion of the  recovery (or delivery) of nicotine to that of codeine phosphate.  And then codeine was perfused through the probe which was immersed in nicotine solution with different concentrations to calculate the proportion, too.  In another condition nicotine was dissolved in rat plasma.  The rat plasma protein binding rate was determined by using retrodialysis and internal standard method in vitro.  The results are as follows: the proportion of the    recovery (or delivery) of nicotine to that of codeine phosphate was fairly stable.  The delivery of codeine was independent of nicotine concentration in the external medium.  Protein binding rate determined by retrodialysis was almost the same as that determined by internal standard method.  It suggests that the internal standard method is an effective way in the determination of nicotine recovery and codeine phosphate can be used as the internal standard.