Since the type of glycoside affects the pharmacokinetic profile of the aglycon after oral ingestion of quercetin glycosides, clinical studies on the pharmacokinetics of quercetin glycosides are required. However, a suitable method to determine the concentrations of quercetin phase II metabolites in human plasma and urine is lacking. Therefore, we developed and validated an LC-MS method for the quantitation of conjugated quercetin using relevant reference standards, including hetero-conjugates with glucuronic acid and sulfonic acid (QC-GA/S). Quercetin hetero-conjugates extracted from rat serum were used for the method development, and reference standards were biosynthesized for the quantitation. The use of a solid-phase extraction (SPE) column in a 96 well format enabled high-throughput analysis of up to 96 tests in a day, without compromising recovery and sensitivity. The SPE column with a weak anion exchange group contributed to the high recovery of QC-GA/S. The method was then validated, and its usefulness was confirmed using clinical samples. QC-GA/S was the predominant phase II quercetin metabolite after the ingestion of quercetin glucoside or quercetin supplements. Moreover, the two peaks of QC-GA/S found in human plasma and urine were isomers of QC-7GA/4'S, which has been reported as the predominant peak in rat plasma. If QC-GA/S in plasma is responsible for a physiological activity of quercetin, it is important to determine the concentration of each QC-GA/S isomer.