Abstract Purpose To assess estradiol (E2) and progesterone levels during ovarian stimulation determined by third-generation (Gen III) and second-generation (Gen II) Elecsys® immunoassays. Methods E2 and progesterone concentrations were measured using Elecsys® Gen III and Gen II immunoassays, and progesterone concentrations on the day of ovulation triggering were determined by LC–MS/MS. This was a retrospective, non-interventional study conducted at European tertiary referral infertility clinics in women aged 18–45 years, with a body mass index 18–35 kg/m2, regular menses, and both ovaries. Results Serum samples were obtained from 230 women classified by oocyte retrieval as poor (33.0%; 0–3 oocytes), normal (40.9%; 4–15 oocytes), or high (26.1%; > 15 oocytes) responders. E2 and progesterone levels increased during ovarian stimulation, with greatest increases observed in high responders. Elecsys® Gen III and Gen II assay results were highly correlated for E2 (Pearson’s r = 0.99) and progesterone (r = 0.89); Gen III results were lower than Gen II for both E2 and progesterone. On the day of triggering, Gen III E2 and progesterone levels showed a difference of − 15.0% and − 27.9%, respectively. Progesterone levels (on day of triggering) measured by LC–MS/MS correlated better with Gen III (0.98) than Gen II (0.90). Mean relative differences for Gen III and Gen II assays versus LC–MS/MS were 14.6% and 62.8%, respectively. Conclusion E2 and progesterone levels determined with Elecsys® Gen II and III assays were highly correlated; results were lower for Gen III versus Gen II. Differences observed for progesterone on the day of triggering may be clinically relevant.