Abstract: OBJECTIVE To develop a LC-MS/MS method for the content determination of acetyl-L-carnitine in human plasma, and to study the pharmacokinetics of chloroacetyl acetyl-L-carnitine tablet in healthy volunteers. METHODS Chloroacetyl acetyl-L-carnitine tablet at the single administration of 0.5, 1.0, 1.5 g and multiple administration of 0.5 g were given to healthy volunteers. Blood samples were collected at 0-24 h. The pharmacokinetic parameters were calculated by measuring the absolute concentration of acetyl-L-carnitine in plasma of healthy volunteers after single and multiple administration. Mildronate was used as the internal standard(IS), the plasma samples were treated with methanol to precipitate protein and analyzed by LC-MS/MS. The detection was accomplished with ESI source operated in positive ionization mode, and ion reaction pairs were selected to be m/z 204.3→145.2(acetyl-L-carnitine), and m/z 147.2→58.2(mildronate). Chromatographic separation had been achieved on EC 250/4.6 NUCLEOSIL 100-5CN with methanol-10 mmol·L^-1 ammonium acetate(with 0.1% formic acid)(85:15) as the mobile phase. RESULTS The linear calibration curves were obtained in the entire range (20-3 000 μg·L^-1) for acetyl-L-carnitine(r=0.999 1), inter-and intra-batch RSDs and matrix effect were <15%. The pharmacokinetic parameters of single dose(0.5, 1.0, 1.5 g) were:AUC_0-t(4 181.77±2 473.24)μg·h·L^-1, (6 099.54±1 939.41)μg·h·L^-1 and (8 064.71±3 575.99)μg·h·L^-1; C_max (611.42±270.76)μg·L^-1, (830.92±233.19)μg·L^-1 and (1 004.67±414.95)μg·L^-1; t_1/2z (4.50±2.93)h, (6.25±3.65)h and (5.76±3.94)h. The main pharmacokinetic parameters of multiple doses were:AUC_0-t (13 728.82±6 493.04)μg·h·L^-1; C_max (1 129.00±374.05)μg·L^-1; t_1/2z (8.57±4.42)h. CONCLUSION The method is proved to be accurate, sensitive and specific to study on the pharmacokinetics of acetyl-L-carnitine in healthy volunteers. There is a significant difference in the pharmacokinetic parameters after single and multiple doses of chloroacetyl acetyl-L-carnitine tablets, and there is no difference between the sexes, healthy subjects are well tolerated.