Background: Levonorgestrel (LNG) is a commonly used emergency contraceptive which can effect sex hormone levels in female blood. The objective of this study was to develop and validate a specific and sensitive LC-MS/MS method for monitoring endogenous sex hormone levels (17 beta-estradiol and progesterone) in female plasma after administration of LNG. Results & methodology: The method was developed, optimized and validated according to the EMA guideline. Assay validation met all the criteria, including good linearity in the range of 0.1-20 ng/ml for 17 beta-estradiol, as well as 0.2-30 ng/ml for LNG and progesterone. Conclusion: The method has been preliminarily applied to explore the variation trend of sex hormone levels in healthy Chinese female volunteers' plasma after oral administration of 1.5 mg LNG tablet. With LNG absorption into plasma, progesterone and estradiol levels descended rapidly, and maintained at low levels in the onset time.