The mineral separation process flotation is fundamentally relying on hydrophobic interactions, which are still not entirely understood and heavily discussed in literature. Here, various possibilities to determine hydrophobic properties of mineral surfaces in water using the concept of colloidal probe atomic force microscopy are introduced. The method is based on the accepted theories of the hydrophobic effect of hydrophobic surfaces in water. Additionally, the hydrophobic parameters are correlated with microflotation experiments for magnetite and quartz surfaces.