Steroids are a major set of endogenous bioactive compounds. Although increasingly popular, their analysis in biofluids by LC-MS is associated with enduring challenges, such as their low endogenous concentrations or the coexistence of numerous isobaric compounds. Their natural presence in biological matrices complicates their absolute quantification in blood, as the obtention of a blank matrix to establish an external calibration curve is impossible. This protocol describes a strategy for developing an LC-MS/MS method for the extended profiling of steroids in serum and plasma, including as much as 171 target compounds, with the additional absolute quantification of four main steroids (cortisol, testosterone, progesterone, and androstenedione). The proposed sample preparation involves protein precipitation in organic solvents and subsequent filtration of the sample on HLB cartridge. The LC method is developed to resolve most isobaric species thanks to a biphenyl stationary phase. MS detection is performed in multiple reaction monitoring mode with post-column addition of ammonium fluoride to enhance sensitivity. A one-point internal calibration strategy is presented for the absolute quantification of endogenous steroids. The application of this method to the NIST Plasma Reference Material (SRM 1950) led to the identification of 69 distinct endogenous steroids, making it the most comprehensive profiling of these compounds in this reference matrix to date. The quantitative performance of the method is assessed with two certified materials and shows satisfactory precision and trueness.