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Determining the correct localization of post-translational modifications (PTMs) on peptides aids in interpreting their effect on protein function. While most algorithms for this task are available as standalone applications or incorporated into software suites, improving their versatility through access from popular scripting languages facilitates experimentation and incorporation into novel workflows. Here we describe pyAscore, an efficient and versatile implementation of the Ascore algorithm in Python for scoring the localization of user defined PTMs in data dependent mass spectrometry. pyAscore can be used from the command line or imported into Python scripts and accepts standard file formats from popular software tools used in bottom-up proteomics. Access to internal objects for scoring and working with modified peptides adds to the toolbox for working with PTMs in Python. pyAscore and is available as an open source package for Python 3.6+ on all major operating systems and can be found at pyascore.readthedocs.io.