Objective  To compare the assessment of endometrial maturation parameters in endometrial secretion samples obtained by a novel minimally invasive technique with those assessed in tissue biopsies. Design  Prospective study. Setting  University Hospital. Population  Healthy female volunteers attending a gynaecological outpatient clinic. Methods  Endometrial secretion fluid and tissue sampling 5 days after a spontaneous ovulation assessed with ultrasound. Main outcome measures  Progesterone (P) receptor, Ki‐67 expression and the Noyes criteria were used to date endometrial biopsies. In the endometrial fluid samples, glycodelin A (GdA), leukaemia inhibitory factor (LIF) and P levels were analysed, and protein content and electrophoresis patterns were determined. Results  All data were correlated to estradiol (E2) and P serum concentrations. The dating according to histology and immunohistochemical staining patterns correlated significantly with GdA levels (r = 0.376, P =0.048) in endometrial fluid samples as well with serum levels of E2 (r = 0.568, P =0.001) and P (r = 0.408, P =0.023). No correlation was observed between tissue dating and LIF levels and protein content in endometrial fluid samples. Conclusions  The measurement of GdA in endometrial secretion samples may provide a less invasive method for assessing endometrial maturation in potential conception cycles without disrupting implantation.