In non-viral DNA delivery, assessing the cellular trafficking of DNA is crucial to understand how transfection systems interact with the cellular machinery. Using a combination of innovative and established imaging assays and flow cytometry, we characterize DNA delivery in HeLa cells with three different transfection systems: (i) the original protein-based TFAMoplex, using the mitochondrial transcription factor A (TFAM) for DNA compaction, (ii) a modified version of the TFAMoplex incorporating a basic leucine zipper (bZIP) domain, (TFAMoplex-bZIP), and (iii) Lipofectamine. Particularly, DNA cell association, uptake, endosomal rupture, cytoplasmic delivery and reporter gene expression are assessed. TFAMoplex-bZIP mediated delivery of Cy3-labeled DNA shows significantly higher cell association compared to the original TFAMoplex and Lipofectamine (15.6, 6.6. and 0.53 Cy3 objects per cell, respectively). However, the highest DNA internalization efficiency is achieved with Lipofectamine (21.8 % vs. ∼15 % for TFAMoplex-based systems). All transfection systems induce endosomal rupture and the formation of barrier-to-autointegration factor (BAF)-positive nucleoprotein structures, termed BAF clusters. With TFAMoplex-bZIP, BAF clusters are numerous, and significantly more cells show reporter gene expression (46.7 %) compared to Lipofectamine (8.7 %). However, reporter protein intensity is highest with Lipofectamine. The described high-precision characterization tools of DNA delivery might be applied to other non-viral transfection agents to identify their bottlenecks in detail.