Cup horn boosters are miniaturized ultrasound baths that maximize efficiency and precision. The optimization of an ultrasonic-assisted derivatization step by means of a cup horn booster and the determination of estrone, 17beta-estradiol, estriol, 17alpha-ethynyl estradiol and mestranol was developed by GC-MS. Different derivatization reagents and solvents were studied for maximizing the di-derivatization of 17alpha-ethynyl estradiol under ultrasound energy. Only N,O-bis(trimethylsilyl)trifluoroacetamide with 1% of trimethylchlorosilane in pyridine gave satisfactory results and this mixture was further used in the optimization of the ultrasound assisted derivatization. The experiment designs included sonication time (1-10 min), sonication power (20-80%), sonication cycles (1-9), derivatization reagent volume (25-125 microL) and solvent volume (25-125 microL). Once the optimum conditions were fixed, the effect of organic matter and the frequency of the water bath change were studied. Finally, the validation of the analytical method was carried out using spiked natural and synthetic waters. Recoveries (natural (138-70%) and synthetic (112-89%)), the LODs (0.35-1.66 ng/L), and LOQs (1.16-5.52 ng/L) and the precision (0.2-5.3%) of the method were studied. This is the first work in the literature where a cup horn booster is used with the aim of minimizing derivatization time during the determination of estrogenic compounds.