A paramount task in RNA interference research is to decipher the complex biology of cellular effectors, exemplified in humans by four pleiotropic Argonaute proteins (Ago1-Ago4). Here, we exploited DNA family shuffling, a molecular evolution technology, to generate chimeric Ago protein libraries for dissection of intricate phenotypes independently of prior structural knowledge. Through shuffling of human Ago2 and Ago3, we discovered two N-terminal motifs that govern RNA cleavage in concert with the PIWI domain. Structural modeling predicts an impact on protein rigidity and/or RNA-PIWI alignment, suggesting new mechanistic explanations for Ago3's slicing deficiency. Characterization of hybrids including Ago1 and Ago4 solidifies that slicing requires the juxtaposition and combined action of multiple disseminated modules. We also present a Gateway library of codon-optimized chimeras of human Ago1-Ago4 and molecular evolution analysis software as resources for future investigations into RNA interference sequence-structure-function relationships.