We thank Jim Chappell and the PLoS Pathogens reviewers for insightful suggestions and critique of the manuscript.  Peptide-Mediated Cellular Delivery of Oligonucleotide-Based Therapeutics In Vitro: Quantitative Evaluation of Overall Efficacy Employing Easy to Handle Reporter Systems Cellular uptake of therapeutic oligonucleotides and subsequent intracellular trafficking to their target sites represents the major technical hurdle for the biological effectiveness of these potential drugs. Accordingly, laboratories worldwide focus on the development of suitable delivery systems. Among the different available non-viral systems like cationic polymers, cationic liposomes and polymeric nanoparticles, cell-penetrating peptides (CPPs) represent an attractive concept to bypass the problem of poor membrane permeability of these charged macromolecules. While uptake per se in most cases does not represent the main obstacle of nucleic acid delivery in vitro, it becomes increasingly apparent that intracellular trafficking is the bottleneck. As a consequence, in order to optimize a given delivery system, a side-by-side analysis of nucleic acid cargo internalized and the corresponding biological effect is required to determine the overall efficacy. In this review, we will concentrate on peptide-mediated delivery of siRNAs and steric block oligonucleotides and discuss different methods for quantitative assessment of the amount of cargo taken up and how to correlate those numbers with biological effects by applying easy to handle reporter systems. To illustrate current limitations of non-viral nucleic acid delivery systems, we present own data as an example and discuss options of how to enhance trafficking of molecules entrapped in cellular compartments. Oligonucleotide-based strategies which can be used to modulate a vast variety of cellular functions represent a promising alternative to conventional therapies (for a review see: [1, 2] ). Among the different oligonucleotides with therapeutic potential are aptamers, transcription factor-binding decoy oligonucleotides, ribozymes, triplex-forming oligonucleotides (TFO), immunostimulatory CpG motifs, antisense oligonucleotides, small interfering RNAs (siRNAs) and antagomirs. Nowadays, these potential macromolecular drugs are generally either relatively easily derived by rational design (e.g. antisense or siRNA) or straightforward selection processes (e.g. aptamers). One of their main advantages over protein-or peptide-based approaches comprises the high specificity for their target while being non-immunogenic. However, despite these advances, a major impediment to the development of nucleic acid-based strategies for treatment and prevention of diseases is the relatively inefficient means to effectively deliver these macromolecules into the desired target cells. Although viral vectors have been widely used to transfer genetic material into cells [3, 4] , they bear an inherent risk for the patient to encounter severe immunological responses or even develop cancer [5] [6] [7] [8] . As a result of these problems much attention has been paid in recent years to the development of non-viral delivery systems. This conception *Address correspondence to this author at the Institut für Molekulare Medizin, Universität zu Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany; Tel: +49-451-500-2745; Fax: +49-451-500-2729; E-mail: restle@imm.uni-luebeck.de includes an assortment of fairly unrelated approaches yielding various degrees of enhanced cellular uptake of nucleic acids. Currently, liposomes and cationic polymers are used as a standard tool to transfect cells in vitro. However, these procedures are characterized by a significant lack of efficiency accompanied by a high level of toxicity rendering them mostly inadequate for in vivo applications. In this context cell-penetrating peptides (see below) represent an interesting alternative as they generally are less toxic than liposomes or cationic polymers. Moreover, they are commonly better suited to transfer cargo into different cell types like non-adherent cells and primary cells, which are hard to transfect using commercially available standard protocols. The most advanced approaches in the field, which are not subject of the present article, are complex carrier systems combining vantages of assorted strategies to generate nanoparticles with better defined properties aimed towards enhanced uptake as well as intracellular trafficking in combination with cell-specific functionalities. For example, there are attempts to combine peptides with cationic liposomes [9] [10] [11] [12] [13] [14] [15] [16] or polyethyleneimine (PEI) [17] . Other strategies are aimed towards the synthesis of high or low molecular weight branched polymers and/or peptides [18] [19] [20] [21] [22] or dendrimers [23, 24] . Even more complex systems are particularly promising with respect to in vivo delivery [25] [26] [27] [28] [29] .