The determination of cell membrane permeability and of the absorption and clearance rates is an important step in the pharmacokinetic profiling of a new drug candidate. In a similar way, research in cell biology relies on the development of cell penetrating biochemical probes allowing to study and interfere with cellular functions and physiology. A variety of methods have been developed to estimate the ability of molecules to enter the intracellular space. They are based either on artificial membranes or on cellular models, with consequently different abilities to mimic the cellular permeation mechanisms. Howev-er, these techniques are often laborious, expensive and have to be combined to get a reliable characterization of the properties of the molecule of interest. This thesis introduces a new strategy to evaluate real-time absorption and clearance kinet-ics of different classes of molecules in living cells. The approach is based on the use of in-tracellular single-chain FRET-based biosensors with a versatile modular design. First, a proof-of-concept bioprobe for inhibitors of the enzyme human carbonic anhydrase II (HCAII) was developed and optimized for intracellular use: the cell-entry dynamics of a selection of well-known sulfonamide drugs was compared and monitored in real time. Then, we adapted the design of the sensory protein to generate a label-free biosensor for inhibitors of the p53-HDM2 interaction, a highly studied protein-protein interaction in cancer biology. The per-meability and the kinetics of cell entry and washout of various underivatized small molecule and peptidic inhibitors could be measured. This thesis represents a step towards the introduction of a general approach for a low-cost, rapid and minimally invasive evaluation of molecular permeabilities in the cell type of choice. Their modular design lends itself to be adapted to the detection of other families of molecules, provided that a binding protein for the molecule of interest is known.