In the past forty years advancements in fluorescence-based methods including imaging (e.g. confocal and multi-photon) and quantitative spectroscopies (e.g. Fluorescence Correlation Spectroscopy) have been applied to systems ranging from solutions to in vivo models: such methods possess the ability to monitor fluorescence intensity fluctuations and offer the potential to unravel biophysical and biochemical phenomena. A major disadvantage associated with these methods is their ever-increasing cost resulting in the development of image analysis tools that offer the potential to exploit hidden information contained in confocal images.The hypothesis pertaining to this thesis is that image analysis tools developed in recent years exemplified by Raster Image Correlation Spectroscopy (RICS), Spatial Intensity Distribution Analysis (SpIDA) and Fluorescence Intensity Gaussian Mixture Model Analysis (FIGMMA) will provide a new insight into current pharmaceutical problems. The application of these methods to the quantification of protein aggregation, monomer/dimer equilibria, p-glycoprotein efflux activity and transcytosis are presented in this thesis.Protein aggregation poses a major challenge to the biotechnology industry which currently lacks analytical capabilities to profile broad particle size ranges. An in-house RICS (ManICS) software was validated against Dynamic Light Scattering and Fluorescence Correlation Spectroscopy (FCS) to determine Bovine Serum Albumin (BSA) aggregate population distributions under accelerated stability conditions. Initial stages implicated in the growth of aggregates are vital to the mechanistic assessment of protein aggregation. Hence, real-time in situ examination of monomer loss and aggregation of BSA was performed at 50 °C to enable continuous assessment with imaging and subsequent SpIDA analysis. Results obtained from this study suggested reversible fluctuation between monomers and dimers for up to four hours.To correlate membrane receptor and transporter expression with activity and enable the comparison of expression in multiple cell lines, population densities of p-glycoprotein transporters and transferrin receptors were determined using SpIDA in samples subjected to immunofluorescence labelling.The Calcein retention assay is a routine approach to determining multidrug resistance associated with p-glcoprotein efflux and the traditional plate reader approach omits microscopic aspects of p-glycoprotein Calcein-AM uptake and efflux. Confocal microscopy and data obtained from image analyses supported the subcellular and intercellular assessment of Calcein accumulation in MDR1-transfected and control cell lines as a function of time and verapamil concentration. Finally, live cell imaging of transferrin vesicular transport and Cell TraceTM Calcein red-orange AM internalisation in combination with traditional Transwell® assays were assessed to compare their transcellular transport and intracellular concentrations in multiple cell lines. Images obtained enabled visualisation of internalisation and following analysis using SpIDA, RICS and FIGMMA the number of intracellular vesicles and dynamic parameters of Cell TraceTM Calcein red-orange diffusion and intracellular concentration were determined.In conclusion, image analysis tools were applied to providing new parametric insights into a number of pharmaceutically-relevant processes and in some instances this is the first example of such studies. Despite current phenomenal advances in image acquisition capabilities, there remains a broad scope for the validation of image analysis tools and their application to a multitude of areas of interest to pharmaceutical and biomolecular research.