Chewing betel quid is a common habit in Taiwan and associated with the risk of oral cancer. Betel quid contains arecoline and arecaidine, which may serve as the exposure biomarkers of a chewing habit. We developed a liquid chromatography-tandem mass spectrometry method for the quantitative analysis of blood arecoline and arecaidine. Because 8-hydroxydeoxyguanosine (8-OH-dG) level in urine is only one early health marker of carcinogenesis, we also examined its association with chewing habit. We found a significant positive correlation between the quantities of betel quid used before the day of drawing blood and arecoline [(Spearman correlation coefficient (r) = 0.81; p value < 0.01) or arecaidine levels (r = 0.86; p value < 0.01)]. Habitual use quantity (quids/day) showed moderate correlation with both arecoline (r = 0.52; p value < 0.05) and arecaidine concentrations (r = 0.51; p value < 0.05). However, there were no significant correlations between total chewing years and concentrations of arecoline and arecaidine in serum or 8-OH-dG in urine. In conclusion, serum arecoline and arecaidine levels are measurable and good indicators for recent betel quid use.