Genetically encoded fluorescent biosensors are useful tools for tracking target analytes in cells, tissues and living organisms. These probes are often chimeric proteins consisting of a recognition element (e.g., a ligand-binding protein) and a reporter element (one or more fluorescent proteins). The analyte-induced conformational change in the recognition element leads to an observable change in fluorescence in the reporter element. Expression of biosensors is noninvasive and can be targeted to specific tissues and cell types using specific promoter and enhancer sequences, and to subcellular compartments with signal peptides and retention tags. Recent improvements in both indicator engineering and microscopy methods enable chronic in vivo measurements. Here, we describe methods used in the design, testing, optimization and application of genetically encoded biosensors, with a particular focus on the widely utilized calcium indicator GCaMP.