Updated project metadata. Investigation of protein phosphorylation in humans has largely focussed on serine (Ser), threonine (Thr) and tyrosine (Tyr), with proven roles for these modifications in all aspects of life. However, mounting evidence suggests that phosphorylation of non-canonical amino acids, including histidine and lysine, may also regulate vertebrate biology. Phosphoproteomics workflows used for high throughput characterisation of canonical phosphorylation events are typically incompatible with analysis of non-canonical phosphorylation due to instability under acidic conditions. Consequently, non-canonical phosphorylation remains unexplored. Here, we report an Unbiased Phosphopeptide enrichment strategy based on Strong Anion Exchange (SAX) chromatography (UPAX), for enrichment of acid-labile phosphopeptides for characterisation by mass spectrometry. Using this approach, we identify extensive phosphorylation of histidine, arginine, lysine, aspartate and glutamate on human proteins. Remarkably, the numbers of phosphoaspartate and phosphoglutamate residues combined approach that observed for phosphothreonine, while phosphorylation of the other non-canonical residues are of the order of that observed for phosphotyrosine. We have used a method for assessing and calibrating False Localisation Rate (FLR), demonstrating that using commonly applied score thresholds results in non-negligible estimates of FLR. Our results show that while it is highly challenging to eliminate false localisation fully, due to the wide search space for non-canonical phosphorylation, many hundreds of sites can still be discovered with confidence. Our study reveals the previously unappreciated diversity of protein phosphorylation in human cells, and opens up avenues for exploring non-canonical phosphorylation in any proteome.