Lateral flow immunoassay devices are potential biosensors for point-of-care diagnostics. However, these devices are limited by low analytical sensitivity when coupled with a visual colorimetric signal. Here, we analyzed key parameters related to nucleic acid lateral flow performance and related this to the analytical theory behind lateral flow functionality. In particular, we predicted a set of different physical running methods to optimize the assay signal intensity by ensuring higher binding of the signal molecules to the DNA. We found that a double-run method improved the assay signal intensity on average by approximately 50%, and a fully absorbed conjugate pad method came close to the double-run signal efficacy at high DNA concentrations. Our results demonstrate that even when the signal molecule is supplied in excess, a significant proportion of analytes bind to the detection antibody in a colorless complex. The lowest detection limits achieved were 0.1 picomole for detection using an anti-Cy5 antibody and 0.01 picomole for the detection using an anti-Texas Red antibody. Our double-run method improvement is generic, does not require additional reagents and equipment, reduces assay costs compared to the fully absorbed method, and is applicable to other lateral flow immunoassays.