Serum concentration and urinary excretion of flavonoids after ingestion of Thai fruits including pineapple (Phuket), guava (Pan See Thong), pomelo (Thong Dee) and strawberry were determined by a developed method of liquid chromatography-tandem mass spectroscopy (LC-MS/MS). After overnight fasting and twenty-four hours of flavonoids-free diet, each group of six healthy volunteers (male/female) was given 200 g of edible part of fresh fruit. Serum were collected before and at 2, 4, 6 and 8 hours and urine sample were collected during 0-4, 4-6, 6-8, 8-12, 12-24 hours after fruit ingestion. Flavonoids in serum and urine samples were extracted by solid phase extraction. In order to determine flavonoids metabolites, - glucuronidase and aryl sulfatase were used to hydrolyze serum and urine samples before extraction. The results demonstrated that only glucuronic conjugated metabolites, not free flavonoids, were detected in serum with time to maximum concentration at 6 hours of all recovered flavonoids. After ingestion of pineapple, metabolites of kaempferol, quercetin and myricetin were detected. In case of guava and pomelo, metabolites of luteolin, kaempferol, quercetin and myricetin; and luteolin and kaempferol were found. Only metabolites of quercetin were detected in serum. In urine both glucuronic and sulfate metabolites of flavonoids were detected within 12 hours after fruit ingestion. The most abundant flavonoids after ingestion of pineapple, guava, pomelo and strawberry are kaempferol, quercetin, luteolin and quercetin, respectively; and total amount are 479, 877, 436 and 136 g free flavonoid equivalent. Bioavailability of flavonoids was estimated, it was account for 5-10% for luteolin, kaempferol and myricetin but 10-40% for quercetin. In conclusion, flavonoids from fruits were able to be absorbed, despite extensively metabolism via conjugation. Bioactivities of conjugated-flavonoids in vivo have not been elucidated. However, we may postulate that Thai fruit could be a good source of flavonoids. Pineapple is a source for kaempferol while guava and pomelo could be a good source for quercetin and luteolin, respectively. This study provides information for selection of fruits as a “functional foods” for various groups of consumer.