In humans, steroids play a broad and vital role in regulation of gene expression, secondary sexual characteristics, maturation, reproduction, cardiovascular health, neurological functions, etc., but imbalance in steroid metabolism is also linked to development and progression of many diseases, such as cancer, neurodegenerative diseases, and cardiovascular diseases. Hence, measurement of steroids in biological samples is essential to monitor human health. Currently, there is radioimmunoassay, gas chromatography-mass spectrometry (GC/MS), and liquid chromatography-mass spectrometry (LC-MS) methods developed for steroid measurements in biological samples. However, these methods require elaborate sample preparation procedures and have concerns(s) related to reproducibility, dynamic range, time, costs, and most importantly the total coverage of steroids. Also currently, there is no method available for comprehensive steroid profiling in a single LC-MS run that includes androgens, corticosteroids, progestogens, estrogens, estrogen metabolites, estrogen conjugates, and estrogen-DNA adducts as well as exogenous steroid derivatives. Here, I present a global steroid metabolic profiling method based on liquid-liquid extraction (LLE) followed by ultra performance liquid chromatography (UPLC)-tandem mass spectrometry (MS/MS) for simultaneous measurement of over 100 indigenous as well as exogenous steroids in about 12 min, without derivatization. The method was successfully applied to determine steroid hormone levels in the breast tissue of healthy women. Overall presence of all major classes of steroids as well as estrogen derivatives was detected in breast tissue.