The "UT-GPCR004 CHO-K1 cell line bright-field and fluorescence microscopy and corresponding segmentation ground truth" dataset contains the raw microscopy images, corresponding bright-field Z-stack based contrast-enhanced images and the corresponding manual ground truth for fluorescence and bright-field images as well as ground truth generated from the fluorescence images using either random forest-based Ilastik model or U-Net3 based deep learning model. The dataset also contains the class balanced version of the ground truth.