Objective: Biomarkers associated with cancer screening (CA125, CA15‐3, CA72‐4, total prostate specific antigen [tPSA]) and the monitoring of pregnancy (pregnancy associated plasma protein‐A [PAPP‐A]) were measured during natural and stimulated ovarian cycles in disease‐free non-pregnant women to determine if they could reflect normal events relating to ovulation and/or endometrial changes. Methods: A total of 73 blood samples (10 women) collected throughout the natural menstrual cycle, and 64 blood samples (11 women) taken during stimulated ovarian cycles, were analysed on the Roche Cobas e411 automated analyser. Results: Detectable levels of tPSA were measured in at least one point in the cycle in 6/10 of women in the natural cycle and 10/11 of women in stimulated cycles, and CA72-4 was detected in only 12/21 women tested. Concentrations of CA125, tPSA, CA15‐3 and CA72‐4 showed no significant difference between the natural and stimulated ovarian cycle groups. On average the mean PAPP‐A of the natural group was (2.41±0.58) mIU/L higher than the stimulated group (t=4.10, P< 0.001). CA125 and CA15‐3 results were both significantly influenced by the stage of the cycle (P<0.0001), whereas tPSA and PAPP‐A concentrations revealed no significant changes (P≥0.65). CA72‐4 was not affected by the stage of the cycle nor ovarian stimulation. Conclusion: Ovarian stimulation reduced serum PAPP‐A levels, CA125 and CA15‐3 levels were generally unaffected by ovarian stimulation but displayed cyclical changes throughout both natural and stimulated cycles, whilst tPSA and CA72-4 were not affected by the stage of the cycle or ovarian stimulation.