Kinetoplastid parasites pose a huge global health burden on both humans and livestock. Current treatments are toxic and difficult to administer. With the emergence of drug-resistant organisms, it is essential to identify new therapeutic targets. African trypanosomiasis, or sleeping sickness, is caused by Trypanosoma brucei. This thesis focuses on the characterisation and validation of a potential new drug target in this kinetoplastid parasite. Flap endonucleases are essential enzymes involved in DNA replication and repair. Synthetic constructs, encoding wild type and mutated FEN genes, were produced and expressed in Escherichia coli. Purification procedures and quantitative enzyme activity assays were developed. The catalytic and binding activity of the D183K variant were determined and compared to the wild type enzyme. The former was shown to be catalytically inert, yet able to bind a DNA substrate with similar affinity to the wild type enzyme (Kd = 2.22 and 1.37 μM respectively). The D183K variant was crystallised collaboratively and its structure determined by colleagues in the Department of Molecular Biology & Biotechnology. The wild type and D183K variant genes were over-expressed in T. brucei bloodstream form cells, using a tetracycline-inducible system. Expression of the catalytically inert protein had a severely detrimental effect on cell growth, and morphological changes were observed 72 hours post-induction. Knock-down assays were attempted using siRNA techniques, but results were inconclusive. The recombinant wild type enzyme was targeted with known FEN inhibitors. Myricetin, a commercially available flavonoid, inhibited T. brucei flap endonuclease (IC50 = 14.81 μM). T. brucei bloodstream form cells were killed upon exposure to myricetin (LD50 = 23.63 μM). A high-throughput thermal shift assay was developed to aid fragment-based drug design. This assay can be used to detect binding by myricetin, a known TbFEN inhibitor. Use of this assay will open up further avenues of exploration for targeting the T. brucei flap endonuclease.