Immunoaffinity chromatography (IAC), mass spectrometry and especially tandem mass spectrometry (MS/MS) represent the most efficient and reliable analytical techniques for specific isolation, unequivocal identification and accurate quantification of numerous natural and synthetic substances in biological samples. This review article focuses on the combined use of these outstanding methodologies in basic and clinical research and in life sciences for the quantitative analysis of low- and high-molecular mass biomarkers, drugs and toxins in urine, plasma or serum samples, in tissue and other biologicals systems published in the last decade. The analytes discussed in some detail include the biomarkers of oxidative stress 15( S)-8- iso-prostaglandin F 2α {15( S)-8- iso-PGF 2α} and 3-nitrotyrosine, the major urinary metabolite of the lipid mediators cysteinyl leukotrienes, i.e., the leukotriene E 4 (LTE 4), melatonin, and the major collagen type II neoepitope peptide in human urine.