Prostate Cancer (PCa) is one of the leading medical issues faced by men worldwide and is the most prevalent cancer diagnosed in men in both Europe and the United States. Some prostatic cancers present as an indolent disease with no clinical symptoms during the lifetime of the patient, and in many cases, men die with the disease rather than from it (insignificant PCa). However, for some PCa patients the disease takes a much more aggressive route, spreading into the seminal vesicles, bladder, and rectum and further metastasising to the lymph nodes, bone and other organs. Measurement of prostate specific antigen (PSA) levels in serum is routinely used for PCa diagnosis and monitoring disease progression (Lilja, H., et al. 1987; J. Clin. Invest 80: 281-285). Conversely, PSA levels can also rise as a result of Benign Prostatic Hyperplasia (BPH) and hence this marker provides little or no insight into the biology of an individual’s prostatic disorder. There is no outright test that can distinguish between significant and insignificant disease. There is an unmet need for better biomarkers to stratify patients into the most appropriate treatment option for their individual prostatic disease. The overall aim of this research was to generate PCa-specific antibodies, namely anti-prostate-specific membrane antigen (PSMA) and anti-secreted frizzled related protein-2 (SFRP2) antibodies, for application in Immunohistochemistry (IHC) to enhance or improve prostate cancer diagnosis. Using a specific targeting strategy, anti-SFRP-2 and anti-PSMA fragment antibodies were successfully produced and characterised. The generation and characterisation approach applied required a combination of the use of a novel antigen expression strategy (for the production of SFRP2), high-throughput assay screening with ‘stateof-the-art’ multiplexed surface-plasmon resonance-based instrumentation and standard analytical techniques. The selected recombinant antibodies were demonstrated to match the performance of current commercially available polyclonal antibodies. In addition, the use of bispecific antibodies for therapeutic and diagnostic application was described and the first step in ‘proof-of-concept’ studies establishing a novel bispecific antibody generation approach is presented.