In this study, we aim at deep mining host-microbial AA-BA metabolites with the aid of mass spectrometry (MS) technique. To this end, we first established a polarityswitching multiple reaction monitoring (MRM) MS scanning method based on liquid chromatography-tandem triple quadrupole mass spectrometry (LC/ESI-QqQ-MS) for AA-BA profiling according to MS fragmentation characteristics of AA-BAs in positive and negative ion modes. This method was then applied for profiling AA-BAs from mouse and human feces, and 118 AA-BA conjugates were successfully screened. Notably, for the first time, 69 AA-BA conjugates involving 14 novel amino acid conjugations with host BAs (serine, valine, threonine, cysteine, asparagine, aspartic acid, lysine, glutamine, glutamic acid, methionine, histidine, arginine, isoleucine, and tryptophan conjugations) were discovered. Moreover, we revealed a significant difference in feces AA-BA content between chronic sleep deprivation (CSD) mice and controls.