“Pharmacological chaperones” are small molecules that are capable of binding to and stabilizing native structures of otherwise folding-defective mutant proteins and thus represent a promising therapeutic approach for many inherited diseases. To date successful pharmacological rescue has been achieved for a small number of targets using known high affinity agonists or antagonists of the protein products of the mutated genes. In most cases, however, the lack of high affinity ligands or structural information for the mutated gene product precludes the use of a rational approach to design ligands that are suitable for pharmacological rescue. Thus, pharmacological chaperone discovery for the majority of targets requires screening of large and diverse compound libraries using high-throughput screening (HTS) technology. We have developed a sensitive and rapid assay to detect folding of conditional mutants of genes encoding membrane or secreted proteins. This assay exploits enzymatic recombination of two fragments of pancreatic ribonuclease and the use of a hypersensitive fluorescent ribonuclease substrate and is well-suited to standard HTS platforms. We will present data demonstrating the efficacy of this approach for several transmembrane and secreted substrates including cystic fibrosis transmembrane conductance regulator (CFTR) and myocilin, a gene linked to hereditary glaucoma.