The Octane Index framework and Co-Optimization of Fuels and Engines (Co-Optima) Central Fuels Hypothesis are evaluated using seven fuels operated under stoichiometric, knock-limited conditions in a directly injected spark ignition (DISI) engine. Of the seven tested fuels, three fuels are “Tier III” fuel blends, meaning that they are blends of molecules which have passed two levels of screening, and have been evaluated to be ready for tests in a research engine. These molecules have been blended into a 4-component gasoline surrogate at varying volume fractions in order to achieve a Research Octane Number (RON) rating of 98. The four molecules under consideration are: isobutanol, 2-butanol, and Diisobutylene. Research engine tests measured knock limits at heated and unheated intake temperature conditions, as well as throttled and boosted intake pressures. The tested knock-limited operating conditions conceptually exist both between the Motor Octane Number (MON) and RON conditions, as well as “beyond RON” conditions. In addition to directly assessing the performance of the Tier III blends relative to other gasolines, the Octane Index framework was evaluated with considerations of experimental uncertainty as well as Research and Motor Octane Number test uncertainties. The Octane Index was found to hold to the first order, although there are deviations from Octane Index behavior which are not likely to be explained by the known experimental uncertainties.