The metabolic fate of anthocyanins until recently was relatively unknown, primarily as a result of their instability at physiological pH and a lack of published methods for isolating and identifying their metabolites from biological samples. The aim of the present work was to establish methods for the extraction and quantification of anthocyanin metabolites present in urine, serum and fecal samples. 35 commercial and 10 synthetic analytes, including both known and predicted human and microbial metabolites of anthocyanins were obtained as reference standards. HPLC and MS/MS conditions were optimized for organic modifier, ionic modifier, mobile phase gradient, flow rate, column type and MS source and compound dependent parameters. The impact of sorbent, solvent, acid, preservative, elution and evaporation on SPE extraction efficiency was also explored. The HPLC-MS/MS method validation demonstrated acceptable linearity (r2, 0.997 ± 0.002) and sensitivity (LODs: urine, 100 ± 375 nM; serum, 104 ± 358 nM and feces 138 ± 344 nM) and the final SPE methods provided recoveries of 88.3 ± 17.8% for urine, 86.5 ± 11.1% for serum and 80.6 ± 20.9% for feces. Final methods were applied to clinical samples derived from an anthocyanin intervention study, where 36 of the 45 modeled metabolites were detected within urine, plasma or faecal samples. The described methods provide suitable versatility for the identification and quantification of an extensive series of anthocyanin metabolites for use in future clinical studies exploring absorption, distribution, metabolism and elimination.