Maternal inheritance of mitochondrial DNA (mtDNA) renders selection blind to mutations whose effects are limited to males. Evolutionary theory predicts this will lead to the accumulation of a male-specific genetic load within the mitochondrial genomes of populations; that is a pool of mutations that negatively affects male, but not female, fitness components. This principle has been termed the Mother’s Curse hypothesis. While the hypothesis has received some empirical support, its relevance to natural populations of metazoans remains unclear, and these ambiguities are compounded by the lack of a clear predictive framework for studies attempting to test Mother’s Curse. Here, we seek to redress this by outlining the core predictions of the hypothesis, as well as the key features of the experimental designs that are required to enable direct testing of the predictions. Our goal is to provide a roadmap for future research seeking to elucidate the evolutionary significance of the Mother’s Curse hypothesis.