In this paper, development and optimization of a new method for determination of the three hormones (estrone, 17-β-estradiol and 17-α-ethinylestradiol) of the Watch list were described. The validated method relies on isotope dilution—two-step solid phase extraction–liquid phase chromatography mass spectrometry method. The measurement procedure validation has been performed in real matrices, including matrix with high suspended particulate matter and high organic carbon contents, to demonstrate its fitness for purpose. Method performances were in agreement with the requirements of the decision 2015/495/EU (maximal acceptable method detection limit and whole-water analysis). Limits of quantification of the method were of 0.4 ng L−1 for estrone and 17-β-estradiol and 0.1 ng L−1 for 17-α-ethinylestradiol. Expanded uncertainties (k = 2) at limit of quantification were equal to 35 % for estrone and 17-β-estradiol and 50 % for 17-α-ethinylestradiol. The method was successfully implemented to monitor French inland surface waters contamination. The survey reveals a chronic state of contamination by estrone (96 % quantification rate) and significant one by 17-β-estradiol and 17-α-ethinylestradiol (15 % quantification rate). Maximum measured concentrations are above predicted no-effect concentrations, indicating potential risk toward the environment.