Hitherto, several aptamers have been selected against cell surface molecules. The use of these aptamers for in vivo applications requires the prior in-depth in vitro evaluation of cell specific binding. Here, we demonstrate the in vitro tests, which are imperatively necessary to evaluate aptamers prior to in vivo applications. Exemplarily, the target binding of a chemically synthesized model aptamer containing phosphorothioate linkages was tested after the induction of the target protein expression on the cell surface by using flow cytometry. Furthermore, different cell types were used to compare the binding of the aptamer. Different single stranded DNA oligonucleotides were selected as negative controls to evaluate sequence specific binding of the aptamer to the cells. In further experiments, the aptamer binding to the target cells was determined in a mixture containing human plasma and peripheral blood cells to simulate the binding of the aptamer to target cells in human whole blood. In this study, we demonstrated the compelling necessity of the in vitro binding tests with the selected aptamers using target and non-target cells, the use of appropriate nonsense aptamers to validate the sequence specific binding of aptamers, and the evaluation of target binding in human plasma containing blood proteins and cells. Thus, we recommend the use of described methods to validate the target specific binding of newly selected aptamers prior to in vivo applications.