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In spite of demonstrated lack of accuracy and consistency, quantification of steroid hormones is still most commonly executed via immunoassays. Mass spectrometric methods with triple quadrupole instruments are well established and, because of their proven robustness and sensitivity, best suited for targeted analysis. However, recent studies have shown that high-resolution mass spectrometers, like quadrupole time-of-flight instruments (QTOF), show comparable performance in terms of quantification and can generate additional sample information via untargeted profiling workflows. We demonstrate that adequate accuracy and selectivity for estradiol and testosterone can be achieved with a QTOF by data-independent acquisition with sequential window acquisition of all theoretical fragment-ion mass spectra (SWATH). Besides potential combination of targeted quantification and untargeted profiling, SWATH offers advantages with respect to sensitivity because the reduced total number of MS/MS experiments could be used to increase accumulation time without increasing cycle time. By applying a surrogate calibrant method leading to successful validation, a reliable method for absolute steroid quantification and high potential for steroid profiling has been developed. Linear calibration was achieved in the range from 10 to 1,000 pg mL−1 for 13C3-estradiol and from 20 to 15,000 pg mL−1 for 13C3-testosterone. Results for inter-day precision (13C3-estradiol: 4.5–10.2%; 13C3-testosterone: 5.1–7.8%) and inter-day accuracy (13C3-estradiol: 94.6–112.8%; 13C3-testosterone: 98.2–107.7%) were found to be well acceptable. Eventually, the method has been utilized to measure clinical samples of a study in which male volunteers obtained transdermal estradiol patches and sex hormone levels were quantified in plasma.