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AbstractDeveloping a universal drug delivery vehicle for sparingly soluble medications remains a challenge, with the most suited vehicles being ionic liquid and surfactant-based microemulsions. Microemulsions formed by two or more immiscible liquids stabilized by surfactants have been a subject of immense importance due to their potential applications in various fields. This study aimed to formulate a microemulsion, which comprises ionic liquid and surfactant, to investigate its potential toward the poorly water-soluble drug rifampicin. The formation and changes in the microstructure of IL-Tween-20 based microemulsion in the presence of a drug (rifampicin) have been established by using UV–visible, fluorescence, and FTIR spectroscopy. The hydrophobic optical/solvatochromic probes pyrene and p-nitroaniline have been used to observe the presence of ionic liquid-rich and bicontinuous microstructures. The incorporation of the drug into the microemulsion and its stability has been evaluated using pH and conductivity (σ) studies. In addition, the particle size analysis and optical microscopic images of the system help in understanding the presence of the drug in the structure of ionic liquid-in-water microemulsion. Lastly, the probes used can be valuable in locating the position of rifampicin, loaded in microemulsion. [ABSTRACT FROM AUTHOR]