The enzyme CaaX geranylgeranyltransferase-I (GGTase-I) post-translationally attaches a 20-carbon isoprenyl lipid (geranylgeranyl) to the cysteine of the canonical CaaX motif at the C terminus of its target proteins. These CaaX proteins are involved in various cell growth and development processes and have roles in human cancers. The canonical Ca1a2X motif is described as C - cysteine, a1a2 - usually aliphatic, X - leucine or sometimes phenylalanine, isoleucine, methionine, or valine. CaaX modifications are commonly believed to increase the target proteins' hydrophobicity and membrane attachment properties. This thesis investigated the hypothesis that GGTase-I targets can deviate from their canonical consensus motif in light of recent evidence indicating that CaaX farnesyltransferase has broader specificity. A comprehensive genetic screen involving a thermosensitive reporter was performed to evaluate yeast GGTase-I activity toward all 8000 possible Cxxx permutations. Orthogonal methods were used to validate subsets of hits from this high throughput screen. These approaches included a small-scale genetic screen using a distinct reporter critical for cell viability and biochemical-based approaches. Our results establish that yeast GGTase-I efficiently targets near canonical- looking sequences (CxaL/F/I/M/V) where the a1 position has considerable flexibility, the a2 position is restricted to a small set of aliphatic residues, and the terminal position is one of a few hydrophobic residues. We have also developed a yeast system expressing human GGTase-I and characterized its target specificity, which is similar to that of the yeast enzyme. We propose that our system can be utilized to assess the target specificity of GGTase-I from other organisms to understand the therapeutic applications of targeting these enzymes.