Immiscible liquid-liquid systems are widely found in nature and industrial processes, covering a variety of applications, including bacterial adhesion, biofilm formation, emulsification, drug delivery, water flooding in oil recovery, and remediation of chlorinated solvents in groundwater. These systems occur when two liquids cannot form a homogeneous mixture due to differences in their molecular properties, such as polarity or density. As a result, the liquids maintain an interface where they come into contact but do not mix, forming distinct layers. This behaviour is influenced by interfacial tension, a force that minimises the contact area between the two liquids and thus exerts a significant influence on the stability and behaviour of the system. Surfactants, or surface-active agents (SSAs), are often used to manage and manipulate the properties of these interfaces. These compounds significantly reduce the interfacial tension between two immiscible liquids. This study aims to experimentally measure the interfacial tension in a liquid-liquid system through a dynamic in situ method using the Rheo-Microscopy apparatus. This equipment allows real-time observation and measurement of fluid interface behaviour under various conditions. In this way, interfacial tension is determined based on existing drop deformation theories, such as the deformed drop retraction method (DDRM). We investigated the interfacial tension behaviour in a system composed of a 95 wt.percent polydimethylsiloxane (PDMS) hexadecane mixture, an 80 wt.percent glycerol solution in deionized water, and the lipophilic non-ionic surfactant Tween 80, with concentrations ranging from 0.0005 to 0.0500 wt.percent.