Protein microarrays represent a powerful technology with the potential to serve as tools for the detection of a broad range of analytes in numerous applications such as diagnostics, drug development, food safety, and environmental monitoring. So far, especially fundamental studies in molecular and cell biology have been conducted using protein microarrays, while the potential for clinical and industrial applications is not yet fully exploited. Key features of analytical protein microarrays include high throughput and relatively low costs due to minimal reagent consumption, multiplexing, fast kinetics and hence measurements, and the possibility of functional integration. Still, to date the technology is primarily used in research laboratories due to some technical hurdles and a lack of approved standards. Issues that need significant improvement to make the technology more attractive for the diagnostic market are for instance: too low sensitivity and deficiency in reproducibility, inadequate analysis time, lack of automation and portable instruments, and cost of instruments necessary for chip production and read-out. The scope of the thesis at hand was to solve some of these problems. Main achievements reported herein are: the introduction of a combined assay format for the simultaneous measurement of high and low abundant analytes applicable for automated measurements; protocols for reduced assay times; the optimization of assays in complex biological fluids achieving high sensitivity; the evaluation and characterization of surface chemistries on glass, metal and polymeric supports for the integration into a biochip; and the characterization and integration of nanostructures manufactured by nanoimprint lithography for application in plasmon enhanced fluorescence read-out. Based on the improvements in sensitivity and analysis time achieved herein further chip developments in the diagnostic field will be pursued.