Line profiler’s purpose is to evaluate datasets in a biological/biophysical context. The software recognizes line shaped structures in image data and computes their mean position and orientation with sub-pixel accuracy. For each line a mean intensity profile is calculated. Utilising the whole image the software prevents biases, caused by preselecting data subsets. A detailed description can be found on readthedocs. The source code as well as a user guide are available on Github.