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The development of a non-invasive method for measuring the internal fluid behavior and dynamics of microchannels in microfluidics poses critical challenges to biological research, such as understanding the impact of wall shear stress (WSS) in the growth of a bone-forming osteoblast. This study used the General Defocus Particle Tracking (GDPT) technique to develop a non-invasive method for quantifying the fluid velocity profile and calculated the WSS within a microfluidic chip. The GDPT estimates particle motion in a three-dimensional space by analyzing two-dimensional images and video captured using a single camera. However, without a lens to introduce aberration, GDPT is prone to error in estimating the displacement direction for out-of-focus particles, and without knowing the exact refractive indices, the scaling from estimated values to physical units is inaccurate. The proposed approach addresses both challenges by using theoretical knowledge on laminar flow and integrating results obtained from multiple analyses. The proposed approach was validated using computational fluid dynamics (CFD) simulations and experimental video of a microfluidic chip that can generate different WSS levels under steady-state flow conditions. By comparing the CFD and GDPT velocity profiles, it was found that the Mean Pearson Correlation Coefficient is 0.77 (max = 0.90) and the Mean Intraclass Correlation Coefficient is 0.66 (max = 0.82). The densitometry analysis of osteoblast cells cultured on the designed microfluidic chip for four days revealed that the cell proliferation rate correlates positively with the measured WSS values. The proposed analysis can be applied to quantify the laminar flow in microfluidic chip experiments without specialized equipment. [ABSTRACT FROM AUTHOR]