Nanoparticle hydrophobicity is a key factor controlling the stability, adhesion, and transport of nanoparticle suspensions. Although a number of approaches have been presented for evaluating nanoparticle hydrophobicity, these methods are difficult to apply to larger nanoparticles and viruses (>100 nm in size) that are of increasing importance in drug delivery and gene therapy. This study investigated the use of a new analytical hydrophobic interaction chromatography method employing a 5.0 μm pore size polyvinylidene fluoride membrane as the stationary-phase in membrane hydrophobic interaction chromatography (MHIC). Experimental data obtained using a series of model proteins were in good agreement with literature values for the hydrophobicity (both experimental and computational). MHIC was then used to evaluate the hydrophobicity of a variety of nanoparticles, including a live attenuated viral vaccine, both in water and in the presence of different surfactants. This new method can be implemented on any liquid chromatography system, run times are typically <20 min, and the experiments avoid the use of organic solvents that could alter the structure of many biological nanoparticles.