Abstract The variable data for pyridostigmine levels reported in human plasma would indicate procedural and/or analytical problems. Consequently an effort was made to establish a precise and sensitive method which could be applied to the quantitation of pyridostigmine in plasma of human as well as other mammalian species. In this method the effect of various types of anticoagulants, the efficacy and precision of the isolation cartridge, the stability of the drug in blood, and the effect of cryogenic storage on the isolation and quantitation of both drug and internal standard were examined. Data on spiked human, guinea pig, dog, and rat plasma at nanogram level of pyridostigmine were presented. Variable concentrations of mobile phase, ranging from 20–27% acetonitrile in water, were used to minimize interfering peaks present in blank samples. Limits of sensitivity in ng/ml (CV) were: human, 2.5 (12.5%), guinea pig, 5.0 (4.5%), dog, 5.0 (2.7%), and rat, 5.0 (7.2%).