Liquid chromatography-tandem mass spectrometry (LC-MS/MS) provides sensitivity and specificity for monitoring tacrolimus drug level in blood, but it requires an LC system and sample preparation, which is not amenable to random access testing typical of immunoassays. Paper spray (PS) ionization generates gas phase analyte ions directly from dried blood spots without sample preparation and LC. We evaluated a PS-MS/MS method for tacrolimus drug monitoring in a clinical diagnostic laboratory. Whole blood sample was mixed with stable isotope labeled internal standard ([(13)C, (2)H2]-FK506) and spotted onto a cartridge containing triangular shaped card paper. After drying, samples were analyzed by PS MS/MS in the selected reaction monitoring (SRM) mode, with a run time of 3 min/sample. Analytical measurement range was 1.5-30 ng/ml. Assay inter-day imprecision was 13%, 8%, and 5% at tacrolimus concentrations of 4.5, 10.5, and 24.5 ng/ml, respectively. Accuracy was determined by pure tacrolimus solution and was confirmed by result correlation to an immunoassay (slope=1.0, intercept=-0.02; r(2)=0.99), and to a conventional LC-MS/MS method (slope=0.90, intercept=0.4; r(2)=0.94). PS-MS/MS provides accurate results for tacrolimus with rapid turnaround time amenable to random access testing protocols.