Cellular traction forces (CTFs) are generated by adherent cells and involved in regulating migration, morphology, and homeostasis. Accurate measurement of CTFs is crucial for understanding fundamental biological processes such as morphogenesis, angiogenesis, and wound healing. However, directly measuring CTFs, which typically range in the nanonewton scale, is challenging. Cellular traction force microscopy (TFM) has been developed to quantify CTFs, but detailed operational procedures and complex data analysis limit its applicability. In this study, hydrogels embedded with fluo-spheres serve as the substrate for TFM measurement under a detailed TFM protocol. Additionally, we designed a user-friendly program for easy parameter setting. An open-source program called Python Fourier transform traction cytometry (pyFTTC) is introduced for data analysis, utilizing particle image velocimetry (PIV) to calculate the traction force from a batch of images. Cross-correlation based PIV and L2-regularized FTTC are applied to all images for data analysis. This article provides a straightforward protocol for quantifying CTFs in standard laboratories, facilitating both cell biology studies and biomaterials development.