The clinical use of poorly water-soluble drugs has become a big challenge in pharmaceutical development due to the compromised bioavailability of the drugs in vivo. Nanocrystals have been proposed as a formulation strategy to improve the dissolution properties of these drugs. The reduction of the particle size down to the nano-sized range has dramatically changed the physicochemical properties of drugs. Drug nanocrystals show particle sizes varying from 100 nm to 400 nm, and can be produced by top-down, bottom-up or via a combination of these techniques. The core of the nanocrystals consists of pure drug, which is covered by a layer of a suitable surfactant. The benefits of using nanocrystals in drug delivery, when compared to other nanoparticles, are related to their production facility, simple structure, and suitability for a variety of administration routes. The greatest disadvantage of nanocrystals is their inherent instability, due to the risk of crystal growth (a process so-called Ostwald ripening). Thus, the selection of an appropriate stabilizer is crucial to obtain long-term physicochemically stable nanocrystals. High pressure homogenization is the most promising production process, which can employed at low or high temperatures. This technique have advantages, including the possibilities of scaling up, lack of organic solvents and the production of small particles diameter with low polydispersity index, usually below 0.2. The sequential use of high shear homogenization followed by high pressure homogenization, can modulate nanoparticles size for different routes of administration. The present study focuses on the optimization of the production process of two formulations composed with different surfactants, and produced by High Shear Homogenization and High Pressure Homogenization. To build up the surface response charts, a 22 full factorial design experiment, based on 2 independent variables, was used to obtain an optimized formulations. The effects of the production process on the mean particle size, polydispersity index were investigated. The in vitro ibuprofen release from the optimized formulations were determined using Franz diffusion cells. Cell viability was assessed for the formulations and different controls on human epithelial colorectal cells (Caco-2). Evaluation of cell viability was performed by a colorimetric assay, i.e., AlamarBlue® assay. The cell viability assay was performed based on the cell capacity to metabolize resazurin, at pre- determined time-intervals, 3, 6 and 24 hours. In both formulations, Caco-2 cells viability was shown to be dependent, both on the drug concentration and time of exposure.