|In this study, a robust, sensitive, reliable and fast method is presented for the quantitation of major steroids in blood using a single LC-MS/MS method with simultaneous positive and negative ionization. LC-MS/MS analysis was performed on a QQQ with ESI positive/negative ion fast polarity switching mode. Major target steroids were estrone, estradiol, estriol, cortisol, progesterone, testosterone, dehydroepiandrosterone sulfate and dehydroepiandrosterone. The reveres phase column with sub 2 µm particles was used. Blood serum or plasma samples were analyzed. All steroids are baseline separated. The LODs and LOQs were evaluated. All calibration curves showed good linearity with over four orders of dynamic range.