Aptamers are short oligonucleotides (< 100 bases) selected from large combinatorial pools of sequences (from 1012 to 1015) for their capacity to bind a target (amino acids, antibiotic, proteins…). Such ligands are isolated by a method of directed molecular evolution usually named SELEX (Systematic Evolution of Ligands by Exponential enrichment). Since 2005, our group and others have adapted the SELEX against whole living cells. Using this strategy, we selected several nuclease resistant 2′-Fluoro-pyrimidines (2′-F-Py) RNA aptamers against cell surface biomarkers that can represent surrogate markers of cancer cells (1-3). This technique, usually named cell-SELEX, allows the parallel selection of several aptamers against membrane proteins in a native conformation. However, although these aptamers are very important tools for in vitro experiments, their translation for in vivo uses is still difficult to predict. To solve this problem, we have developed fluorescence imaging techniques to investigate their biodistribution and tumour targeting capacities in small animal models (4-5). Particularly, we demonstrated that fluorescence diffuse optical tomography (fDOT) can reach nanomolar sensitivity, even in a deep-seated organ (5). Hence, six nuclease resistant 2′-fluoro-pyrimidine (2′-F-Py) RNA aptamers identified from cell-SELEX were screened in nude mouse xenograft models. Despite high affinity against cells in vitro, only one aptamer (named ACE8) exhibited significant tumor uptake compared to a control sequence (0.67 ± 0.16% of injected dose 3 hours after injection compared to 0.07 ± 0.06%, respectively). This aptamer was then used to purify and identify its target, annexin A2 (AII), a known cancer biomarker involved in metastasis and angiogenesis.