Microfluidic chips in the last two decades have had significant advances in the analysis of INTERFACIAL tension phenomenon due to their many advantages. To analyze INTERFACIAL tension phenomena, droplet flow in microchannels can be used. In this study, water-n-hexane INTERFACIAL tension in the presence of surface-active agents was measured, using microfluidic tensiometry. For this purpose, a glass microfluidic flow-focusing junction was fabricated for generating n-hexane droplets within an aqueous phase. The dependence of droplet size on the concentration of surfactants has been investigated. A theoretical equation was developed, considering force balance on the droplet generation in the microfluidic device, giving a relation between the INTERFACIAL tension and the generated droplet sizes. By standardizing the microfluidic chips with the aid of a system, whose INTERFACIAL tension is known (hexane normal and tween 20 in distilled water), INTERFACIAL tension can be measured with measuring the size of produced droplets for other systems that can form droplets in the microchannel. In this study, the microfluidic device and the relation were employed to measure the INTERFACIAL tension in the presence of either of sodium dodecyl sulphate (SDS) or Cetyl trimethylammonium bromide (CTAB) surfactants. It was found that the measured INTERFACIAL tensions deviate less than 10% compared to those measured with a commercially available ring method.