In the fields of diagnosis, imaging, regenerative medicine, and drug targeting, aptamers are promising nucleic acid ligands for specific recognition and binding of whole living cells. These aptamers are selected by a combinatorial chemistry technique called cell-SELEX (Systematic Evolution of Ligands by EXponential enrichment). During this iterative procedure of in vitro selection and enzymatic amplification, the enrichment of cell binding aptamers is generally monitored by flow cytometry. This method needs the use of fluorophore-labeled oligonucleotides for detection and allows only the relative evaluation of the aptamer binding compared with the control. Here, we describe the development and validation of a new quantitative real time polymerase chain reaction (qPCR) method for the absolute determination of cell bound aptamers during cell-SELEX. The method is based on SYBR Green I real-time PCR technology and uses an aptamer standard curve to determine the accurate aptamer amount on cells after the incubations. Lysates of cells with bound aptamers were used to identify the absolute amount of aptamers on cells. This method is highly sensitive and allows the detection of very small quantities of aptamers in cell lysate samples. The lower detection limit is 20 fg. The established qPCR method can be used as an additional monitoring tool during cell-SELEX to determine the enrichment of cell binding aptamers on cells, whereby the absolute quantity is determined. Furthermore, the contamination of the amplified aptamer pool with by-products can be prevented by prior determination of bound aptamer amount on cells.