AIM: To investigate the changes of anesthetic drug concentration in plasma during isolation of autologous blood with acute normovolemic hemodilution and its influence on the depth of anesthesia, muscle relaxant effect and blood drug concentration after reinfusion. METHODS: Forty patients of both sexes, aged 20-60 yr, American Society of Anesthesiologists physical status ⅠorⅡ, hemoglobin (Hb) >120 g/L, hematocrit (Hct) >35%,undergoing eletive multilevel spinal surgery were included, were divided into 2 groups (n=20 each) using a random number table. ANH group (group A): ANH was performed after stable induction of anesthesia, the target Hct value was 28%-30%, and autologous blood was reinfused after the main operation steps. Control group (group C): routine transfusion and infusion treatment. The bispectral index (BIS) and Train-of-Four stimulation (TOF) were observed and recorded at the stable induction of anesthesia (T1), 30 minutes of stable induction (T2), the end of operation (T3), 30 minutes after the end of the operation (T4), 1 hour after the end of the operation (T5) and 2 hours after the end of the operation (T6). The concentrations of propofol and cisatracurium besylate in plasma at T1-T6, stored blood at 1 h (TS1), 2 h(TS2), and before reinfusion (TS3) were detected by Liquid Chromatography- tandem Mass Spectrometry. The extubation time and recovery score at T4-6 hours were recorded. RE- SULTS: There was no significant difference in propofol between the two groups at each time point (P>0.05). The plasma concentration of cisatracurium besylate in group A was higher than that in group C at T3 (P<0.05). The concentration of two kinds of anesthetic drugs in blood samples decreased slightly with time,but there was no significant difference between groups (P>0.05). The BIS value at T4 and TOF value at T3 in group A were significantly lower than those in group C. The recovery score of group A was lower than that of group C at T4 (P<0.05). There was no significant difference in extubation time (P>0.05). CONCLUSION: The plasma concentrations of propofol and cisatracurium besylate were basically unchanged during the in vitro isolation of ANH autologous blood. The plasma concentrations of cisatracurium besylate were only temporarily affected after the main operation steps, but the postoperative muscle relaxation recovery and recovery quality were not significantly affected.