Background: Nucleofection is an emerging technology for delivery of nucleic acids into both the cytoplasm and nucleus of eukaryotic cells with high efficiency. This makes it an ideal technology for gene delivery and siRNA applications. A 96-well format has recently been made available for highthroughput nucleofection, however conditions must be optimized for delivery into each specific cell type. Screening each 96-well plate can be expensive, and descriptions of methods and outcomes to determine the best conditions are lacking in the literature. Here we employ simple methods, including cell counting, microscopy, viability and cytotoxicity assays to describe the minimal experimental methods required to optimize nucleofection conditions for a given cell line. Methods: We comprehensively measured and analyzed the outcomes of the 96-well nucleofection of pmaxGFP plasmids encoding green fluorescent protein (GFP) into the A-549 human lung epithelial cell line. Fluorescent microscopy and a plate reader were used to respectively observe and quantify green fluorescence in both whole and lysed cells. Cell viability was determined by direct counting/permeability assays, and by both absorbance and fluorescence-based plate reader cytotoxicity assays. Finally, an optimal nucleofection condition was used to deliver siRNA and gene specific knock-down was demonstrated. Results: GFP fluorescence among conditions ranged from non-existent to bright, based upon the fluorescent microscopy and plate reader results. Correlation between direct counting of cells and plate-based cytotoxicity assays were from R = .81 to R = .88, depending on the assay. Correlation between the GFP fluorescence of lysed and unlysed cells was high, ranging from R = .91 to R = .97. Finally, delivery of a pooled sample of siRNAs targeting the gene relA using an optimized nucleofection condition resulted in a 70–95% knock down of the gene over 48 h with 90–97% cell viability. Conclusion: Our results show the optimal 96-well nucleofection conditions for the widely-used human cell line, A-549. We describe simple, effective methods for determining optimal conditions with high confidence, providing a useful road map for other laboratories planning optimization of specific cell lines or primary cells. Our analysis of outcomes suggests the need to only measure unlysed, whole-cell fluorescence and cell metabolic activity using a plate reader cytotoxicity assay to determine the best conditions for 96-well nucleofection.