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The Organ-on-a-Chip is a microfluidic device designed to replicate human organ and tissue conditions. Temperature regulation is crucial for mimicking physiological processes and ensuring cellular viability in Organ-on-a-Chip platforms. This article focuses on employing system identification to mathematically model thermal regulation in a new Organ-on-a-Chip. A controlled environment box was constructed for testing, using infrared and negative temperature coeficient sensors for intrim temperature analysis. Data collected from the cell culture chamber were processed and analyzed in MATLAB®. System identification led to the selection of a transfer function, and a descriptive equation for Organ-on-a-Chip temperature was obtained. The study revealed temperature differences between ambient and culture chamber temperatures, emphasizing the importance of real-time analysis. Mathematical modeling and in silico tests play a crucial role in predicting physiological behavior, allowing complex interactions to be simulated. The obtained temperature equation can be refined and applied in various 3D models. indicating innovation in the integration of Organ-on-a-Chip in medicine and biomedical engineering. [ABSTRACT FROM AUTHOR]