Background: The major limitation of blood hormone measurements is the rapid daily concentration fluctuations. However, hair hormone measurement may provide an information on chronic body hormone exposure. Objective: The aim of the current study is to determine concentration of estradiol (E) and progesterone (P) in scalp hair of pre and postmenopausal women, using the widely used commercial immunoassay, which is also in use to detect serum E and P in most laboratories. Materials and Methods: The study included women under age of 40 years with regular cycles, and postmenopausal women over the age of 50 years, with no cycles for at least five years. Following hair processing, concentration of E and P in hair was assayed by chemiluminescent enzyme immunoassay. Results: The study included 22 premenopausal women (mean age of 30±4.8 years), and 17 postmenopausal women, (mean age of 62 ± 8.1 years), Mean E and P concentrations were higher in premenopausal women compared to postmenopausal women; however, it did not reach statistical significance [8.6 (3.3-16.4) vs. 5.9 (4.3-8.7) pmol/gr-hair (p = 0.53) and 0.006 ([0.004-0.011) vs. 0.005 (0.003-0.006) pmol/gr-hair (p = 0.10), respectively). Conclusion: Hair E and P concentrations can be quantified by immunoassay but it may not be the optimal method. Future studies should be conducted in order to determine the optimal technique in measurement of these hormones from scalp hair.