Characterizing the thermal behavior of dispersed droplets within microfluidic channels is crucial for different applications in lab-on-a-chip. In this paper, the physics of droplets volume during their transport over a heater is studied experimentally and numerically. The response of droplets to external heating is examined at temperature ranges of 25-90 °C and at different flow rates of the dispersed phase respect to the continuous flow. The results present a reliable prediction of the droplet volume and stability when heating is applied to the droplets at the downstream channel in a quite far distance from the droplets' ejection orifice. Increasing the ratio of flow rate resulted in larger droplets; for instance, the flow ratio of 0.25 produced drops with 40% larger diameter than the flow rate of 0.1. For every 10 °C increase in temperature of the droplets, the droplet diameter increased by about 5.7% and 4.2% for pure oil and oil with a surfactant, respectively. Also, the droplets showed a degree of instability during their transport over the heater at higher temperatures. Adding SPAN 20 surfactant improved the stability of the droplets at temperatures higher than 60 °C. The experimentally validated numerical model helped for systemic analysis of the influence of key temperature-dependence parameters (e.g. surface tension, density and viscosity of both phases) on controlling the volume and stability of droplets. Our findings supported to develop highly functional systems with a predetermined droplets performance under high temperatures up to 90 °C. This report provides a preliminary basis for enhancing the performance of droplet microfluidic systems for digital droplet polymerase chain reaction (ddPCR), continuous flow digital loop-mediated isothermal PCR (LAMP), and droplet-based antibiotic susceptibility testing.