Flavonoids and their derivatives are known for their diverse biological activities. This study aims to elucidate the structure-activity relationships (SARs) of flavonoids, including fisetin, luteolin, quercetin, and mono-methylated quercetins (MQs), with a focus on their potential as therapeutic agents for colorectal cancer (CRC). Using electrospray ionization tandem mass spectrometry (ESI-QTOF MS/MS) and retro Diels-Alder (rDA) analysis, we developed a novel analytical method to differentiate between MQs, despite their identical molecular weights, by analyzing their unique fragmentation patterns. Comparing the structures and activities of the tested flavonoids highlights the importance of the methylation and hydroxylation status at the carbon 3, 5, 7, 3', and 4' positions of quercetin for enhancing antiproliferative activity in human CRC cells. Specifically, 3-O-methylquercetin and 4'-O-methylquercetin were found to induce cell cycle arrest and apoptosis in CRC cells through mechanisms involving oxidative stress, mitochondrial dysfunction, and inactivation of the SRC/JAK2/STAT3 pathway, while exhibiting no cytotoxicity to normal human colon cells. These results suggest that MQs are promising therapeutic flavonoids for CRC treatment. This study underscores the importance of specific structural modifications in flavonoids to improve their anticancer efficacy, providing valuable insights for the development of targeted therapies for CRC.