Selective isolation of biologically important molecules and their functional characterization is one of the primary goals of bioanalytical chemistry. Several different affinity tools such as antibodies, affimers, nanobodies, and DARPins have been explored to achieve these goals. In recent years, oligonucleotide based affinity tools called aptamers have become progressively attractive and the research in this area has seen an exponential increase. Aptamer probes have been explored in many different areas of bioanalytical chemistry such as electrochemical and optical biosensor development, targeted drug delivery, logic gates, DNA nanotechnology, and point of care diagnostics. However aptamers are still largely overlooked in mass spectrometry (MS) and biomarker discovery. After the completion of the human genome project, the focus has shifted towards functional genomics and to understand the living systems by deciphering the functions of proteins and metabolites. Therefore identification and functional characterization of these molecules are of outmost importance. Although identification of isolated biomolecules and analysis of simple biological mixtures using MS have become relatively simple, the power of MS gradually decreases as the complexity of biological mixtures increases. Therefore the development of selective and targeted approaches is at the forefront of mass spectrometry. Aptamers have great potential in affinity mass spectrometry to improve selectivity, specificity and throughput. Herein, bioanalytical mass spectrometry and biomarker discovery applications of aptamers will be reviewed.