Nowadays, fluorescent protein (FP) variants have been engineered to fluoresce in all different colors; to display photoswitchable, or photochromic, behavior; or to show yet other beneficial properties that enable or enhance a still growing set of new fluorescence spectroscopy and microcopy techniques. This has allowed the (in situ) study of biomolecules with unprecedented resolution, specificity, sensitivity, and ease of labeling. However, brighter FPs, more photostable FPs, and FPs that display an even better compatibility with biophysical microspectroscopic techniques are still highly desired. The key characteristics of FPs-absorption spectrum, emission spectrum, brightness, fluorescence lifetime, maturation rate, oligomeric state, photostability, pH sensitivity, and functionality in protein fusions-determine their application. This chapter will describe these key features and present several experimental protocols to optimize them.The optimization procedure contains three steps. First the amino acid sequence of a template FP is changed via random or site-directed mutagenesis. A primary screening based on fluorescence intensity, fluorescence lifetime, and emission spectrum is applied on the FP libraries expressed in bacteria. The most promising mutants are isolated, purified, and characterized in vitro. In this step all key characteristics are determined experimentally. Finally the new FPs are evaluated for use in vivo. The protein production and maturation is monitored in bacteria, while transfected mammalian cells report on the photostability, relative brightness, and correct localization to various subcellular compartments.