Several countries have set ambitious targets for the transport sector that mandate a gradual increase in advanced biofuel content in the coming years. The current work addresses this transition and indicates two promising gasoline bio-blendstocks: anisole and isobutanol. The whole value chains of these bio-components were considered, focusing on end-use performance, but also analyz-ing feedstock and its conversion, well-to wheel (WTW) greenhouse gas (GHG) emissions and costs. Three alternative fuels, namely a ternary blend (15% anisole, 15% isobutanol, 70% fossil gasoline on an energy basis) and two binary blends (15% anisole with fossil gasoline and 30% isobutanol with fossil gasoline), were tested, focusing on their drop-in applicability in spark ignition (SI) engines. The formulated liquid fuels performed well and showed the potential to increase brake thermal efficiency (BTE) by 1.4% on average. Measured unburned hydrocarbons (HC) and carbon monoxide (CO) emissions were increased on average by 12–29% and 17–51%, respectively. However, HC and CO concentrations and exhaust temperatures were at acceptable levels for proper catalyst operation. The studied blends were estimated to bring 11–22% of WTW GHG emission reductions compared to base gasoline. Additionally, the fleet performance and benefits of flexi-fuel vehicles (FFV) were modeled for ternary blends.