In this study, we developed a new liquid chromatography–mass spectrometry (LC–MS) method for analysis of the protoberberine alkaloid palmatine and its metabolites with separation performed on a cyanopropyl-modified stationary phase. Palmatine (10 μM) was metabolized using suspensions of human hepatocytes and human recombinant cytochrome P450 (CYP) enzymes. Our analyses using electrospray ionization-quadrupole time-of-flight mass spectrometry revealed that palmatine was relatively resistant to the metabolic activity of human hepatocytes and recombinant CYP enzymes. However, we found that the biotransformation of palmatine in human hepatocytes included O-demethylation or hydroxylation, and that the product of palmatine demethylation was conjugated by glucuronidation or sulfation. Moreover, we found that human recombinant CYP2D6 and, to a lesser extent, CYP1A2 can mediate O-demethylation of palmatine. These results provide fundamental insights into the biotransformation of palmatine in human in vitro models and, together with the LC–MS method, can be applied for further studies on the biotransformation of palmatine and other protoberberine alkaloids.