The retinoblastoma tumor suppressor (Rb) is a multifunctional protein that primarily regulates the cell cycle but also has roles in cellular differentiation, DNA damage response, and apoptosis. The loss of Rb is a key event in the development or progression of many cancers. Essential functions of Rb occur through its pocket domain, which is necessary for regulating binding interactions with E2F transcription factors and transcription repressors that bind via an LxCxE motif. The pocket domain is the most highly conserved region of the multidomain protein, as well as the most frequent site of mutations. To understand what effects cancer missense mutations have on Rb's pocket domain, we used fluorescence polarization and differential scanning fluorimetry to quantify changes caused by 75 cancer-associated missense variants to E2F transactivation domain (E2F