The development of delivery systems for therapeutic oligonucleotides is a fast growing field. Owing to the enormous potential of short nucleic acids as alternative drugs such a growth is not unexpected. Besides viral vectors there is a highly diverse and constantly increasing number of non-viral systems evolving. However, despite considerable progress achieved in recent years, even the most advanced systems either lack the efficiencies required for downstream drug development or do show a substantial degree of toxicity or both. Of the many factors which limit their use, cellular uptake of the cargo/carrier complexes and subsequent intracellular trafficking to reach the target site are the most important. In addition to such essential considerations there are various additional parameters to be taken into account like serum stability, pharmacokinetic features and tissue barriers as well as target cell specificity. Now the question arises where to start optimizing a given delivery system. Currently, there is a clear trend towards in vivo testing. In principle such a development is a step in the right direction since many of the experimental data derived from artificial cell tissue culture systems with established cell lines are not applicable to the in vivo situation. On the other hand, it is questionable to what extent such animal experiments will eventually pay off as long as important fundamental problems remain largely unsolved. As outlined above, our quantitative studies along with microscopic analyses of siRNAs and steric block oligonucleotides using either a peptide or a commercially availably cationic lipid as carrier clearly show that less than 0.1% -5% of molecules taken up are involved in a biological response, i.e. RNAimediated down regulation or splice correction-mediated up regulation of reporter gene activity. Evidently, the vast majority of internalized cargo never reaches the target. This implies that uptake per se is not the limiting factor here. Although there are no such detailed quantitative numbers available in the literature for other systems, there are countless reports showing that to various degrees this applies to almost any non-viral delivery approach currently available. Taken together, if one would succeed to optimize intracellular trafficking this holds the potential to boost overall efficacy by up to 3 orders of magnitude. Moreover, it is reasonable to assume that such fundamental cellular restrictions can be adequately investigated in tissue culture without the use of animal models. So it might be worthwhile to reconsider the concept of maybe premature in vivo testing by moving backwards one step and first optimizing the systems with regard to intracellular limitations before dealing with the next level of complexity. Accordingly, in vitro model systems like the ones described above for siRNAs or steric block oligonucleotides are valuable tools to study particular aspects of nucleic acid delivery. However, currently available data are based on studies using a variety of different cell lines and techniques, which renders a direct comparison of different delivery approaches impossible. In this context it would be highly desirable to introduce standardized protocols for in vitro testing (e.g. the splice correction system developed by Kole and coworkers [121] ) together with methods for detailed quantitative analyses (e.g. the liquid hybridization protocol [55, 176] ). This would facilitate a direct quantitative comparison of exceedingly diverse approaches on at least the cellular level.