A selective, sensitive and rapid mice dried blood spot (DBS) method has been developed and validated for the simultaneous quantification of bendamustine (BM) and γ-hydroxy-bendamustine (HBM) as per regulatory guidelines using an LC-MS/MS. Quality control, calibration curve and study sample DBS cards were sonicated with 5% formic acid in water before extraction with ethyl acetate enriched with internal standard (I.S.). The organic layer was evaporated and residue was reconstituted in 0.1% formic acid in acetonitrile for LC-MS/MS analysis. Chromatographic resolution of both analytes (BM and HBM) and the I.S. (loperamide) was achieved on an Atlantis dC18 column using 0.2% formic acid:acetonitrile (25:75, v/v) as an eluant delivered at a constant flow-rate of 0.5 mL/min. The total chromatographic run time was 3.2 min. The MS/MS ion transitions monitored were m/z 358.0 → 228.0, 374.0 → 338.0 and 477.0 → 210.0 for BM, HBM and the I.S, respectively. The assay was linear in the range of 5.65-2544 ng/mL for both BM and HBM. The within-run and between-run accuracy and within-run and between-run precision were in the range of 0.96-1.00 and 1.36-9.94%, respectively for BM; 0.88-1.03 and 4.57-11.7%, respectively for HBM on mice DBS cards. Stability studies showed that both analytes were stable at room temperature for 7 days and at -80 °C for 55 days on DBS cards. The validated DBS method has been applied to a pharmacokinetic study in mice.