Continued success in drug discovery largely relies on the development and evolution of assay technologies capable of accurate representation of cellular behaviour in response to specific stimulations. The development of such technologies is vital to the drug discovery process as it guides the selection of promising compounds as well as the early abandonment of potential failing drug candidates. Compared with the data obtained using cell-free assays, direct monitoring of drug-modulated signalling in live cell systems offers high content information, in conditions designed to closely resemble a physiological environment. These benefits have driven the use of whole cell systems in drugs screening and fundamental biomedical research. In cell-based assays, compounds are either screened on genetically modified cells expressing an exogenous receptor or on selected primary cells expressing the target of interest. In this context, a compound can simultaneously behave as an agonist or antagonist depending on which intracellular signalling pathway is monitored. Therefore, a lack of efficacy 354for a preselected biochemical signalling event does not necessarily mean a lack of receptor activation. Thus, because of the ability of ligands to stabilize or stimulate subsets of receptor activities, a receptor activation screen should not rely on a single specific assay, but rather on an integrated approach to measure multiple signalling events simultaneously. Unlike label-dependent cell assays that measure specific cellular events, cell-based biosensing technologies such as those based on impedance mesurement 13 or evanescent field at surface 45 have the potential to provide an integrated cellular response. The recognition of this need for more in-depth analysis of biological activity beyond simple specificity, selectivity and affinity is clear. Hence, what is required is a high content information on the action of molecules on drug targets and, especially, a more precise profile of the molecular pathways induced by a particular candidate drug molecule. In this chapter, we present an experimental strategy based on atomic force microscope (AFM) force sensing on individual cells to report on the effects of external pharmacological stimuli on cellular functions. This kind of cell-based biosensing allows for label-free, multimodal and real-time monitoring of cellular responses and signalling pathways in recombinant cell models as well as primary cell cultures related to physiologically and pathophysiological models. Exposition of receptors present at the external surface of cells to external chemical or biochemical messenger entities (hormones, neurotransmitters, ions, light, scent, taste, etc) leads to the activation of a variety of intracellular molecular signalling pathways which are often associated with change in cell morphology, cell motility or specific proteins expression. Here we show that AFM-based force measurements in conjunction with fluorescence imaging of intracellular component can fingerprint the contribution of signalling pathways subsequent to the activation of specific receptor at the cell membrane.