Endocrine-disrupting chemicals (EDCs) are compounds that interfere with hormonal system and the most commonly studied EDCs are those that affect reproduction. A class of particularly potent ECDs are the natural and synthetic steroid hormones. For example, recent estimates of the predicted-no-effect concentration (PNECs) of estradiol, which has the strongest physiological activity in estrogen, was about 0.35ng/L on fish spices. In comparison, the PNEC of the well-known ECD, bisphenol A, was estimated 60ng/L. So that owing to the low effect and occurrence level of steroid hormone analytical method are needed. Here, we demonstrates a complete method to rapidly and precisely determine five steroid hormones, estrone (E1), estradiol (E2), estriol (E3), progesterone, and testosterone, in milk with Agilent 6495 Triple Quadrupole Mass Spectrometer coupled with Agilent 1290 Infinity II UHPLC. Quantitation was achieved with sample QuEChERS procedure without any derivatization.