Blood orange juice has a high content in anthocyanins, especially represented by delphinidin-3-glucoside (D3G), cyanidin-3-glucoside (C3G) and cyanidin-3-(6-malonylglucoside) (CMG). An LC-MS/MS method for the simultaneous determination of D3G and C3G in human plasma and urine was developed and validated. After sample preparation by SPE, chromatographic separation was performed with an RP-C(18) column, using a water/methanol linear gradient. The quantitation of target compounds was determined by multiple reaction monitoring (MRM) mode, using ESI. The method showed good selectivity, sensitivity (LOD = 0.05 and 0.10 ng/mL for C3G in plasma and urine, respectively; LOD = 0.10 ng/mL for D3G in plasma and urine), linearity (0.20-200 ng/mL; r >or= 0.998), intra- and interday precision and accuracy (<or= 14.6%) and satisfactory recoveries (80.0-110.4%) for the target compounds in both plasma and urine. Stability of analytes in plasma and urine has been investigated in detail. This method was successfully applied to the determination of D3G, C3G and CMG levels in human plasma and urine after the ingestion of a single dose (500 mL) of blood orange juice.