A new,rapid and sensitive method was developed for the quantifying of rhodamine 123 (R123) in rat plasma using liquid chromatography tandem mass spectrometry (LC-MS/MS).Rhodamine 6G (R6G) was used as the internal standard (IS).R123 and IS were extracted from aliquots of plasma with ethyl acetate and dichloromethane (4∶1) as the solvent.Chromatographic separation was performed using a Zorbax Eclipse Plus C18 column.The mobile phase was composed of A:ammonium formate-formic acid buffer containing 5 mM ammonium formate and 0.1% formic acid and B:methanol (A∶B,5∶95,v/v).To quantify R123 and IS respectively,multiple reaction monitoring (MRM) transition of m/z 345.2 →285.2 and m/z 443.3 →415.2 were performed.The analysis lasted for 4 min,in the positive mode,the results of which were presented as a linear calibration curve over the concentration range of 1-200 ng/ml and sampling volume of 2 μl.The lowest limit of quantification (LLOQ)reached 1 ng/ml.The intra and inter-day precision were 6.7% and 9.2% for the low quality control (QC) sample (2 ng/ml),respectively.Intra and inter-day precision of the medium QC sample (100 ng/ml) were both less than 3.4%.Intra and inter-day precision of high QC sample (150 ng/ml) were both less than 2.1%,while the intra and inter-day relative errors ranged between-7.4% and 9.1% for the above three QC concentration levels.The LC-MS/MS method proved to be simple,accurate,reliable and with a shorter running time and has been successfully applied to an absorption experiment in the rat.