Poor aqueous solubility and moderate permeability of Nelfinavir mesylate (NFM) leads to high variability in absorption after oral administration. To improve the solubility and bioavailability of NFM, the self microemulsifying drug delivery system (SMEDDS) was developed. For this purpose, Quality by design (QbD) approach employing D-optimal mixture design was used to prepare SMEDDS of NFM. Further, the software generated numerically optimized SMEDDS were developed by utilizing desirability function. Maisine 35-1, Tween 80, and Transcutol HP were identified as oil, surfactant, and co-surfactant that had best solubility for NFM. Ternary phase diagrams were plotted to identify the self-emulsification region. Dissolution of putative NFM in simulated fasted or fed small intestinal conditions, respectively, predicted that there is a positive food effect. However, NFM loaded SMEDDS showed absence of food effect with no significant difference in dissolution performance either in Fasted or fed state simulated intestinal fluid (FaSSIF or FeSSIF) biorelevent dissolution media. The prepared SMEDDS were thermodynamically stable with droplet size (121 nm), poly dispersity index (PDI) (0.198) and emulsification time (<1 min). Transmission electron microscopy (TEM) analysis confirmed the spherical shape of the reconstituted SMEDDS droplets. The ex vivo performance revealed 4.57 fold enhancement in the apparent permeability of NFM as compared to NFM suspension. The animal pharmacokinetic analysis in New Zealand strain rabbits indicated food effect on pure NFM suspension. However, absence of food effect and 3.5-3.6 fold enhancement in the oral bioavailability was observed when NFM was formulated into SMEDDS. Thus, it could be envisaged that development of SMEDDS formulation of NFM could be one of the best alternative to enhance oral bioavailability of NFM.