Investigation of nanoparticle-mediated nucleic acid delivery is a key step for the development of nucleic acid-based nanotherapeutics. Using luciferases as reporter genes, the efficiency of gene delivery can be quantified in a highly sensitive way based on bioluminescence measurements. Here we describe a robust assay to quantify the activity of exogenously produced firefly luciferase and its normalization by the total protein amount (bicinchoninic acid assay, BCA) in the cells. The method describes preparation of firefly luciferase assay buffer (F-LAB) along with BCA assay and employment of the optimized firefly luciferase assay for investigating in vitro gene delivery by polyplex and lipoplex nanoparticles. Reusability of F-LAB for ease of usage (by freezing and reusing it for luciferase assay) is also demonstrated.