Using UPLC coupled with triple quadrupole mass spectrometry (QqQ-MS) in the multiple reaction monitoring (MRM) mode and HPLC coupled with quadrupole time-of-flight tandem mass spectrometry (Q/TOF-MS) in the MS/MS mode, twelve bile acids in Bile Arisaema were analyzed qualitatively and quantitatively. The fragmentation route of seven bile acids has been postulated. Over a wide concentration range, the quantification technique showed outstanding linearity, repeatability, stability, accuracy, and recovery. Seven chemicals in Bile Arisaema have compositions that varied greatly by area. The fermentation period of bile acid was effectively adjusted using chemometric methods, hierarchical clustering analysis (HCA), and principal components analysis (PCA).