<p>Bile acids (BAs) are endogenous signaling molecules with diverse biological functions. In cholestasis, conjugated BAs are markedly elevated, making their ratio to unconjugated BAs a critical diagnostic biomarker. Accurate quantification of BAs is pivotal for assessing cholestatic disease progression. Conjugated BAs contain a fragile amide bond that is susceptible to hydrolysis in biological samples, resulting in an artificially low ratio of conjugated-to-unconjugated BAs. Here, we systematically investigated the benchtop stability of BAs in murine biological samples using liquid chromatography-mass spectrometry. Strikingly, pronounced degradation of conjugated BAs occurred in liver and ileum samples from wild-type and Mdr2 knockout mice within 1&#xa0;h on the benchtop at 25 ℃. In liver samples, tauro-α/β-muricholic acid (T-α/β-MCA), the predominant murine conjugated BAs, decreased by over 70%. Other conjugated BAs, such as taurochenodeoxycholic acid (TCDCA) and tauroursodeoxycholic acid (TUDCA), also showed significant degradation. Concurrently, unconjugated BAs increased by 1-12-fold. In ileum samples, conjugated BAs exhibited a 5%–40% reduction concomitant with up to a 30-fold increase in unconjugated BAs. In contrast, BAs remained stable in serum samples. Mechanistic studies using deuterium-labeled conjugated BAs confirmed amide bond hydrolysis as the primary degradation pathway. Several optimized protocols, such as immediate storage on ice, enzymatic inactivation, and liquid nitrogen snap-freezing, effectively mitigated the hydrolysis. These findings suggest that the hydrolysis of conjugated BAs in untreated liver and ileum samples leads to serious underestimation of conjugated BAs and inflation of unconjugated BA levels, highlighting a preanalytical pitfall in BA quantification. Stabilizing protocols are essential immediately upon sample collection.</p> Graphical abstract <p></p>