An ultra high performance liquid chromatography tandem mass spectrometry method (UHPLC-MS/MS) was developed for the determination of 33 target and 28 unknown bile acids (BAs) in biological samples. Sixty-one BAs could be measured in 20 min using only a small amount of sample and with a simple sample preparation. The method proved to be very sensitive (limit of detection 5-350 pg/mL, lower limit of quantitation 0.1-2.6 ng/mL), linear (R2?>?0.99) and reproducible (typically CV <15 % in biological matrixes). The method was used to analyze human adipose tissue, plasma, and serum (from same subjects) and mouse serum, gall bladder, small intestine, and colon samples (from same animals). Cholic acid, ursodeoxycholic acid, and chenodeoxycholic acid, deoxycholic acid, and their conjugates (mainly glycine, but also taurine conjugates) were the main metabolites in human samples, and cholic acid, glycine cholic acid, and several taurine conjugates in mouse samples. Using the method, 28 unknown BAs could also be detected. UHPLC-MS/MS spectra, accurate mass, and tissue distribution suggested that nine of the unknown bile acids were taurine conjugates, 13 were glycine conjugates, and six were intact BAs, respectively. To our knowledge, this was the first time BAs were detected in adipose tissue. Results showed that 17 targeted BAs were found at ng/g level in human adipose tissue. Our findings give a novel insight of the endogenous role of BAs in adipose tissue and their role as biomarkers (e.g., in metabolic diseases).