Abstract Background Lenalidomide (LND) is a potent novel thalidomide analog which demonstrated remarkable clinical activity in treatment of multiple myeloma disease via a multiple-pathways mechanism. Validated sensitive method with high throughput is required for the determination of lenalidomide for pharmacokinetics and toxicokinetic studies. Ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) is a preeminent analytical tool for rapid biomedical analysis. Results A simple, highly sensitive UPLC-MS/MS method was developed and validated for the determination of LND in rabbit and human plasma. After a simple protein precipitation using methanol, LND and carbamazepine (IS) were separated on Acquity UPLC BEH™ C₁₈ column (50 × 2.1 mm, i.d. 1.7 μm, Waters, USA) using a mobile phase consisted of acetonitrile:water:formic acid (65:35:0.1%, v/v/v) pumped at a flow rate of 0.2 mL/min. LND and IS were eluted at 0.71 and 1.92 min, respectively. The mass spectrometric determination was carried out using an electrospray interface operated in the positive mode with multiple reaction monitoring (MRM) mode. The precursor to product ion transitions of m/z 260.1 > 149.0 and m/z 237.0 > 179.0 were used to quantify LND and IS, respectively. The method was linear in the concentration range of 0.23–1000 ng/mL with a limit of quantitation of 0.23 ng/mL. All the validation parameters were in the ranges acceptable by the guidelines of analytical method validation. Conclusion The proposed UPLC-MS/MS method is simple, rapid and highly sensitive, and hence it could be reliable for pharmacokinetic and toxicokinetic study in both animals and humans.