Etimicin (ETM), which belongs to the newest generation of aminoglycosides (AGs), has been proven to not only maintain but also strengthen the advantages of former AGs with relatively less toxicity. Now, it is widely applied for the treatment of bacterial infections in the clinic. Nevertheless, nephrotoxicity and ototoxicity are unavoidable issues for AGs, and while ETM is no exception, the seriousness of these issues is different. To explore the reason why ETM exhibits less toxicity and to better direct the optimization and development of new AGs, it is of great necessity and importance to monitor the pharmacokinetic behaviors of ETM in its potential toxicity target organs, the kidney and internal ear, as well as in plasma. Therefore, a novel, sensitive and efficient LC–MS/MS method without derivatization or ion-pairing agents had been developed and validated for quantification of ETM in rat plasma, kidney and internal ear for the first time. This method showed good linearity over the range of 50–2000 ng/mL for rat plasma/internal ear and 100–5000 ng/mL for rat kidney. The precision was less than 4.4% and the accuracy was below 4.8%. Recovery and matrix effects were 71.3%–82.8% and 97.6%–108.5%, respectively. After intravenous administration of a single dose of ETM, plasma drug concentrations fit well with a two-compartmental model, and the AUC0-∞, t1/2α, t1/2β, MRT and CL were 127.96 ± 5.52 μg *h/mL, 0.53 ± 0.03 h, 3.32 ± 1.11 h, 1.01 ± 0.03 h and 234.80 ± 10.05 mL/h/kg, respectively. Particularly, ETM showed a considerably long half-life in kidney and internal ear, up to 155.96 ± 19.95 h and 83.11 ± 26.60 h, respectively, which might contribute greatly to its toxicity.