The ribosome, a 2.5-MDa molecular machine that polymerizes α-amino acids into proteins, is the catalytic workhorse of the translation apparatus. The catalytic capacity of the translation machinery has attracted extensive efforts to repurpose it for novel functions. One key idea is that the natural translation machinery can be harnessed to synthesize polymers containing non-natural building blocks. Expanding the repertoire of ribosomal substrates and functions is a difficult task, however, because the requirement of cell viability severely constrains the alterations that can be made to the ribosome, a catalyst that sustains the life of a cell. These constraints have made the natural ribosome nearly unevolvable and, so far, no generalizable approach for modifying the catalytic peptidyl transferase center (PTC) of the ribosome has been advanced. We address this grand challenge by using an in vitro ribosome synthesis, assembly, and translation system (termed iSAT) that harnesses the biosynthetic potential of cellular machines without using intact cells. Here, we use iSAT to generate variant ribosomes with mutations in the PTC, and inquire how these modifications impact protein synthesis. Using iSAT, we assembled 180 different variant ribosomes possessing single-base substitutions of 23S rRNA nucleotides in the active site. By successfully quantifying full-length protein synthesis kinetics of iSAT-assembled wild type and mutant ribosomes, we found many key PTC mutations, which were expected to abolish ribosomal activity, still permitted full-length protein synthesis. We also assessed translation fidelity and ribosome assembly, as well as mapped mutant activity onto the ribosome’s crystal structure. Our work provides the first and only comprehensive mapping of the impacts of every mutation within the ribosome’s active site on protein synthesis. The understanding gained from these studies facilitates efforts to engineer and evolve ribosomes for synthetic biology.