The immunophilins are a superfamily of proteins comprising the cyclophilins, the FKBPs and the parvulin sub-families. Members are present ubiquitously in plant and animal cells, acting as both prolyl-isomerases and signalling proteins. Some also have chaperone activity. The prolyl isomerase function of the immunophilins has been identified as being central to progression of a large number of diseases, making them tempting drug targets. Whilst there are several assays which can be used to identify inhibitors of the prolyl isomerase function, they are hampered by one or more problems: multistep mechanisms, poor signal-to-noise ratios, expensive, laborious and unamenable to high throughput screening. Multiple fluorescent systems (fluorescence anisotropy, FRET, 2D-FIDA/FCS) and several technologies (solution and solid phase synthesis, solution and solid phase screening, combinatorial synthesis, and stopped-flow spectrometry) were explored to develop a system suitable for fast, efficient screening of immunophilins. The most promising of these is a prototype assay based on the design, cloning, expression and production of fluorescently labelled mutant of cyclophilin B, which shows an increase in fluorescence emission upon cyclosporin ligand binding.