A sensitive and specific liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of indocyanine green (ICG) in dog plasma and bile. An ICG analog (IR-820) was used as an internal standard. A protein precipitation method was used with acetonitrile for plasma sample preparation, whereas bile samples were diluted with water (120-fold) prior to analysis. Using MS/MS in the multiple reaction monitoring mode, ICG and IR-820 were detected in both matrices without interference. The lower limit of quantitation for ICG in dog plasma was 3 ng/mL, with an intra- and inter-day accuracy (%Bias) and precision (%CV) of less than 15%, so it was possible to study the pharmacokinetics of ICG in bile duct-cannulated dogs and assess their liver function after surgery. The method described herein is sensitive, selective and faster than other existing methods (e.g., spectrophotometry, HPLC/UV–vis detection, or HPLC/fluorescence detection).