BACKGROUND: Individuals who are unable to metabolize the short-acting muscle relaxant succinylcholine due to abnormal cholinesterase activity are currently investigated via spectrophotometry using artificial substrates and enzyme inhibitors. Methods have been described using succinylcholine as substrate but with measurement of the product choline. However, choline may be released from other endogenous substrates within the serum. Direct measurement of the in vitro metabolism of succinylcholine as substrate may provide a better indication of the in vivo situation with regard to cholinesterase status. METHODS: The rate of in vitro metabolism of succinylcholine by cholinesterase was measured using liquid chromatography linked to tandem mass spectrometry (LC-MS/MS). A comparison was made using serum samples in which cholinesterase activity had been measured using propionylthiocholine as substrate and phenotyped by enzyme inhibitor studies. RESULTS: A good correlation (r = 0.9, P < 0.0001) was found between cholinesterase activity measured by LC-MS/MS using succinylcholine as substrate compared with propionylthiocholine as substrate measured spectrophotometrically. All serum samples with a cholinesterase activity of <1 IU/L, as measured using succinylcholine as substrate, were considered to be at increased risk of succinylcholine sensitivity. These latter results correlated well to the atypical phenotypes. CONCLUSIONS: A simple and fast LC-MS/MS technique for the measurement of cholinesterase activity using succinylcholine as substrate has been described. This method clearly identifies patients at risk of prolonged apnoea following succinylcholine administration and compares favourably with existing spectrophotometric methods using artificial substrates.