The aim of present study was to develop a rapid and reliable analytical method for quantification of ketamine and its major metabolite, norketamine, in bovine plasma. A cost-effective and high-throughput extraction procedure was combined with a sensitive and specific liquid chromatography-heated electrospray ionization-tandem mass spectrometry method. Sample preparation consisted of a protein precipitation step using methanol followed by a clean-up using a HybridSPE(A (R))-phospholipid column and further dilution by a factor 1/10 with water before injection onto the LC-MS/MS instrument. A gradient elution program was performed with 1 % formic acid in water and 0.1 % acetic acid in methanol as mobile phases. Ionization was performed by a heated electrospray ionization (h-ESI) probe operating in the positive ionization mode. Following transitions were monitored in the selected reaction monitoring mode: 238.0 > 124.9/220.0 for ketamine and 224.0 > 207.0/124.9 for norketamine. The method was validated in-house. Matrix-matched calibration graphs were prepared for ketamine and norketamine and correlation and goodness-of-fit coefficients were 0.9991 and 0.9997 and 7.7 and 4.0 %, respectively. The within- and between-run precision and accuracy were evaluated and the results fell within the ranges specified. The limit of quantification was 0.1 A mu g/mL for both analytes, whereas limits of detection were 9.6 and 3.5 ng/mL for ketamine and norketamine, respectively. The method was applied on biological samples from a pharmacokinetic study in calves and demonstrated the suitability of the method for this application.