To create a basic configuration file that will work for most projects is very simple. I assume your box files for training are in the folder ''train_annot'' and the corresponding images in ''train_image''. I furthermore assume that your box size in your box files is 160. To create the config config_cryolo.json simply run: cryolo_gui.py config config_cryolo.json 160 --train_image_folder train_image --train_annot_folder train_annot To get a full description of all available options type: cryolo_gui.py config -h If you want to specify separate validation folders you can use the %%--%%valid_image_folder and %%--%%valid_annot_folder options: cryolo_gui.py config config_cryolo.json 160 --train_image_folder train_image --train_annot_folder train_annot --valid_image_folder valid_img --valid_annot_folder valid_annot