This paper describes optimization of LC-MS/MS conditions to develop a method for selective and sensitive determination of estrogens, namely: estradiol (E2), estrone (E1), estriol (E3), and synthetic estrogen as ethinyl estradiol (EE2) by using statistical experimental design methods. The optimization studies were conducted in three stages: (i) determination of minimum alkaline volume ratio (0–17% NH₄OH at 0.2 M) in mobile phase to maximize peak area by single factor experimental design; (ii) optimization of LC elution conditions including flow rate, acetonitrile concentration in standard and in mobile phase by Box–Behnken response surface method (RSM), and (iii) optimization of LC-MS/MS conditions for seven factors to maximize peak areas by Box–Behnken RSM. NH₄OH volume ratio significantly affected the peak area and it was maximized at 3–5% volume ratio. Predicted optimal LC elution conditions were % ACN_standard: 28, % ACN_mobile:44 and flow rate of 137 μL min⁻¹. The optimum instrumental conditions were determined as sheath gas pressure:33 arbitrary unit (Arb), ion sweep gas pressure: 0.4 Arb, aux gas pressure: 17 Arb, capillary temperature: 254 °C, vaporizer temperature: 352 °C, collision gas pressure: 1.9 mTorr, and spray voltage: 2740 V. Optimization provided substantial improvements in peak symmetry and resolution factor and a 20–25 times peak signal gain with respect to the instrumental self-optimized condition at detection limits of ng L⁻¹ levels were achieved. The lower detection limits were obtained by coupling the method with a SPE procedure for attaining high pre-concentration factors. The signal enhancement was about three orders of magnitude, which constitutes a remarkable sensitivity of the method.