Reagentless biosensors are single molecular species that report the concentration of a specific target analyte, while having minimal impact on the system being studied. This chapter reviews such biosensors with emphasis on the ones that use fluorescence as readout and can be used for real-time assays of concentration changes with reasonably high time resolution and sensitivity. Reagentless biosensors can be designed with different types of recognition elements, particularly specific binding proteins and nucleic acids, including aptamers. Different ways are described in which a fluorescence signal can be used to report the target concentration. These include the use of single, environmentally sensitive fluorophores; FRET pairs, often used in genetically encoded biosensors; and pairs of identical fluorophores that undergo reversible stacking interactions to change fluorescence intensity. The applications of these biosensors in different types of real-time assays with motor proteins are described together with some specific examples. These encompass regulation and mechanism of motor proteins, using both steady-state assays and single-turnover measurements.