Volumetric absorptive microsampling device (VAMS) was evaluated for bioanalysis of tacrolimus, which was used as a model drug with high blood cell partition. Aliquots of blood (ca. 10 μL) with different hematocrits and fortified with tacrolimus were wicked up by VAMS and tacrolimus was extracted with a methanol-water mixture (1:1, v/v) via sonication. After chromatography on an AQUITY UPLC HSS T3 column (100 × 2.1 i.d., mm, 1.8 μm), tacrolimus and the internal standard ascomycin, were detected in the positive ion mode with electrospray ionization by monitoring of transitions m/z 826.6 → 616.4 and m/z 814.6 → 604.0, respectively. An assay method to quantify tacrolimus from 1 to 250 ng/mL in whole blood was qualified by ensuring that linearity, selectivity, intra- and inter-batch reproducibility, and stability were within the acceptance criteria. Consistent and high extraction recovery of tacrolimus was ensured from blood with low- (20%), mid- (45%), and high-hematocrit (65%) levels with minimal matrix effects. Apparent instability at ambient temperature or 4 °C possibly due to reduced recovery suggests that tacrolimus in VAMS should be stored at −25 °C until assay. Potential reduced recovery over time from VAMS should be taken into consideration in method optimization.