Measurement of serum estradiol (E2) and progesterone (PRG) allows monitoring of follicular growth and prediction of the day of ovulation in natural cycles. In combination with vaginal ultrasonography, serum E2 and PRG are used for the monitoring of ovarian superovulation for assisted reproduction. We carried out a clinical evaluation of the VIDAS E2 and PRG assays on a fully automated random access analyzer (miniVIDAS, bioMérieux) by retrospective analysis of frozen serum samples obtained from 16 natural cycles monitored for frozen embryo transfer (FRET) and in superovulation cycles. The superovulation cycles consisted of 28 long gonadotropin-releasing hormone (GnRH) agonist protocol cycles and 15 GnRH antagonist protocol cycles. In FRET cycles we found a good correlation between serum E2 and the diameter of the dominant follicle measured by ovarian ultrasonography. Serum E2 values measured throughout the cycles and follicular phase PRG values were representative of normo-ovulatory cycles. In GnRH agonist long protocols, we found serum E2 and PRG values compatible with castration levels induced by pituitary desensitization. During gonadotropin treatment a close, highly significant correlation was observed between serum E2 and ultrasonographic follicle measurements with both GnRH analogue protocols. Serum PRG measured for 5 days prior to human chorionic gonadotropin (hCG) administration in the agonist cycles was compatible with the absence of premature luteinization. In conclusion, VIDAS E2 and PRG assays are precise and sensitivetoolsforevaluating natural cycles (based on the results in FRET cycles) and for monitoring ovarian superovulation.