With the economic development, the non-renewable resources consumption and the energy crisis, microbial-based platforms represent a promising alternative route for the production of valuable chemicals. Yarrowia lipolytica is a non-conventional oleaginous yeast, which is able to grow on hydrophobic substrates, such as triglycerides, as carbon source. Its growth is associated with the production of different high-value bio-products, such as enzymes, organic acids, and lipids. In recent years, both basic and applied research have been carried out for improving its genetic manipulation and industrial use. The thorough knowledge of Y. lipolytica biolipid conversion and production pathways makes it a good candidate for its use as a cell factory for the design of alternative bioprocesses based on renewable substrates. Waste cooking oils (WCOs) are vegetable oils and animal fats that are discarded after food processing. Globally, high amounts of this waste product are produced every year. Due to their composition in triglycerides, WCO can be used as feedstock for microbial growth to create novel routes for the so-called “industrial symbiosis” following the circular economy approach, to upgrade a waste as a renewable feedstock for the bio-based industry. In particular, the goal of this study was the creation of new value chains from waste cooking oil (WCO) for the sustainable production of added-value compounds to valorize this type of waste and to reduce its incorrect disposal. Specifically, Y. lipolytica was investigated to evaluate its ability to grow in the presence of different concentrations of WCO compared to glucose as carbon source and determine its response both in terms of industrially-relevant compounds production and cell robustness. Flow cytometry analysis was performed to investigate the response of Y. lipolytica at increasing WCO concentrations in terms of intracellular lipids quantification and cellular viability.