Detection of vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3 and vitamin E is important in neurological diseases such as epilepsy. Traditional HPLC-based methods require labor intensive and time-consuming sample extraction procedures, which limits the applications of these methods. In this work, a high-throughput and robust HPLC-MS/MS method with simple sample preparation for the simultaneous quantification of vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3 and vitamin E in rat plasma was developed. These four fat-soluble vitamins were separated on a Kinetex C18 column (50 × 3.0 mm, 2.6 μm) using gradient elution. The LLOQs of vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3 and vitamin E were 50 ng/ml, 1 ng/ml, 5 ng/ml and 1 μg/ml respectively. The intra-day precision and accuracy were in the range of 1.31–13.0% and 85.8–114% respectively, and the inter-day precision and accuracy were 2.47–12.8% and 86.1–115% respectively. After fully validated, the HPLC-MS/MS method was applied to our pentylenetetrazole (PTZ)-induced kindling rat model.