Abstract Background We have created a software implementation of a published and verified method for assigning probabilities to potential phosphorylation sites on peptides using mass spectrometric data. Our tool, named PhosCalc, determines the number of possible phosphorylation sites and calculates the theoretical masses for the b and y fragment ions of a user-provided peptide sequence. A corresponding user-provided mass spectrum is examined to determine which putative b and y ions have support in the spectrum and a probability score is calculated for each combination of phosphorylation sites. Findings We test the implementation using spectra of phosphopeptides from bovine beta-casein and we compare the results from the implementation to those from manually curated and verified phosphopeptides from our own experiments. We find that the PhosCalc scores are capable of helping a user to identify phosphorylated sites and can remove a bottleneck in high throughput proteomics analyses. Conclusion PhosCalc is available as a web-based interface for examining up to 100 peptides and as a downloadable tool for examining larger numbers of peptides. PhosCalc can be used to speed up identification of phosphorylation sites and can be easily integrated into data handling pipelines making it a very useful tool for those involved in phosphoproteomic research.