A sensitive and specific liquid chromatography tandem mass spectrometry method for quantification of 1-methylpyridinium (1-MP) and 1,4-dimethylpyridinium (1,4-DMP) in rat plasma and tissues homogenates was developed. Chromatographic separation was performed on an Aquasil C18 analytical column with an isocratic elution of acetonitrile and water, both with an addition of formic acid (0.1%, v/v). Detection was achieved by triple quadrupole mass spectrometer TSQ Quantum Ultra equipped with a heated electrospray ionization source (HESI). The limit of quantification for both compounds was 0.05 μg/mL in plasma and 0.25 μg/g in studied tissues. The method was applied to pharmacokinetics and bioavailability of both 1-MP and 1,4-DMP with tissue distribution of 1,4-DMP in rats. Pharmacokinetic studies of 1-MP and 1,4-DMP were carried out following their intravenous or intragastric administration to male Wistar rats at the dose of 100 mg/kg. The terminal half-lives of 1-MP and 1,4-DMP after their intravenous administration were 55.3 and 70.8 min, respectively. The absolute bioavailability was 51 and 31% for 1-MP and 1,4-DMP, respectively.