Background. Compared to dsDNA, single-stranded DNA (ssDNA) offers greater structural flexibility, lower immunogenicity, and is utilized in CRISPR-Cas9 homology-directed repair, recombinase-mediated insertions, molecular probes, and expression vectors. We developed a protocol based on the selective 5′–3′ degradation of 5′-phosphorylated dsDNA, followed by magnetic carboxylated bead capture, to generate and purify µg-scale, transfection-grade ssDNA for immunocompetent mammalian cells. Methods. Input dsDNA was generated via preparative PCR (Q5 DNA polymerase, 5′-phosphorylated primer), purified with AmpliClean™ magnetic beads, and digested with Lambda exo (NEB). ssDNA was purified using Quick-DNA/RNA™ MagBeads (Zymo Research) and verified by agarose gel electrophoresis. Residual dsDNA was quantified via Qubit™ 1X dsDNA HS Assay post-S1 nuclease digestion. Endotoxins were measured using the PyroSmart NextGen® kinetic protocol. Results. To upscale the standard Lambda exo digestion (100 ng/µL, 1 U/µg DNA, 30-min incubation) to a 100 µL reaction with 20 µg of dsDNA, the enzyme-substrate ratio and incubation time were titrated. Complete digestion was achieved at an enzyme-substrate ratio of 1.25 U/µg without degrading the non-phosphorylated strand. Optimal incubation times scaled with fragment length: 2 h (>3500 bp), 1.5 h (>1500 bp), and 1 h (<500 bp). Because Lambda exo digests retained a stable fraction of residual dsDNA, even in an over-digestion regime, enzymatic removal using dsDNA-selective nucleases (ExoIII (NEB) and ezDNase™ (ThermoFisher)) was tested. In all tested conditions, neither nuclease exhibited the required selectivity, causing major ssDNA degradation, likely due to residual secondary structures. We then evaluated TRI Reagent® (Sigma) extraction and Quick-DNA/RNA™ MagBeads to selectively capture ssDNA. While the TRI Reagent® extraction resulted in recovering both ssDNA and dsDNA in an organic phase, Quick-DNA/RNA™ MagBeads successfully isolated pure ssDNA in the unbound fraction (recapitulating RNA behavior). The protocol was tested at various dsDNA loads, confirming robust performance up to 20 µg of initial mass load. Processed ssDNA maintained homogeneity during all processing stages, yielding <2% residual dsDNA carryover and endotoxins <0.08 EU/µg in a final prep at end-to-end yields of 33–52% (for the longest and the shortest fragments, respectively). Conclusions. Our optimized Lambda exo protocol effectively scales ssDNA production across diverse fragment sizes. Combined with Quick-DNA/RNA™ MagBeads purification, it reliably yields homogeneous, low-dsDNA and endotoxin-free ssDNA, suitable for mammalian cell transfection.