Background: The Bile-processed Rhizoma coptidis (BRC), which has a colder drug property than Rhizoma coptidis (RC), is widely used for the treatment of heat syndrome. We compared the pharmacokinetics of the protoberberine-type alkaloids in BRC and RC in rats with heat syndrome to elucidate the bile-processing mechanism. Material and Methods: We established a rapid and sensitive method for simultaneously determining three alkaloids: berberine, palmatine, and jatrorrhizine, in rat plasma based on ultra-performance liquid chromatography/tandem mass spectrometry. The separation was carried out on a Waters ACQUITY BEA C18 column. The mobile phase consisted of acetonitrile (containing 0.1% formic acid) and water (containing 0.1% formic acid and 10 mmol/L ammonium acetate) and carbamazepine was used as an internal standard. The detection was carried out in a multiple reaction monitoring mode (MRM) using electrospray ionization in the positive ion mode. Results: Pharmacokinetic profiles indicated that the Cmax of berberine and palmatine increased two times and the Tmax of the three alkaloids decreased three times after bile processing. AUC0→∞ and AUC0→t of the alkaloids were similar between RC and BRC. Conclusion: The results suggest that bile processing could increase the absorption rate of alkaloids. This study broadens our understanding of Chinese herbal medicine processing.