With the aim to investigate the changes of steroid hormones in the serum and gonadal tissue of Japanese eel(Anguilla japonica) during artificial induction of maturation,we have developed and validated a UPLC-MS/MS method to determine 6 steroids in serum samples simultaneously, including testosterone(T),estrone(E₁),17α-hydroxyprogesterone(17α-OHP),17α, 20β-dihydroxy-4-pregnen-3-one(DHP),estradiol-17β(E₂),estriol(E₃),and 4 steroids (T, DHP, E₂, E₃) in gonadal tissue. The results showed that the average recoveries in serum and gonadal tissue detection were 86.9%-98% and 75.6%-88.1%,respectively; and the detection limit(S/N>10) were 0.1ng/g and 0.1 ng/mL. The steroid hormones in serum and gonad tissue of experimental groups were increased through the hormone injection. Except for the content of E₃, all the other five steroid hormones reached their highest content during spawning, however,the E₂ and DHP in gonadal tissue of experimental groups reached the highest content during dystocia. Conversely, not any of E₁and E₃ in serum, as well as E₂ and E₃ in gonadal tissue were detected through the period in the control group. These results provide preliminary understanding of sex steroid hormones changes during the maturation of Anguilla japonica,moreover,it established a sensitive and stable method to determine the sex steroid hormones in aquatic animals.