The low sensitivity of traditional LFIA assays has limited their use in applications where detection of low analyte concentrations is crucial. To address this limitation, researchers have proposed the use of soluble wax barriers to selectively and temporarily accumulate target and label nanoparticles on top of the test line (TL). This extended incubation step promotes the formation of the immune-complex, resulting in a 51.7-fold sensitivity enhancement and up to 96% signal enhancement for Human IgG (H-IgG) detection, compared to the conventional LFA. In this study, a LFA modified with time-delay barriers was developed as a strategy for sensitivity and signal enhancement. The modified assay achieved an inner incubation time of applicability with immunoglobulin depleted human serum. The proposed method represents a promising solution to improve the analytical performance of LFAs without sacrificing their ease-of-use and low cost. Moreover, this approach could be beneficial in applications where high sensitivity is required, or bioreceptors with lower affinity or nucleic-acid based probes are used, as they may require more interaction time with the target analyte.