Engineering proteins through chemical or genetic manipulations offers great potential to design and study proteins with complex properties. Despite tremendous progress, the type and combination of chemical modification (e.g. non-canonical amino acids (ncAAs) or post-translational modifications (PTMs)) that can be incorporated by genetic means (e.g. amber codon suppression) is still limited. Semi-synthetic approaches offer a post-translational alternative to synthetically introduce modifications into proteins, but have typically been performed in vitro and restricted to small proteins that can be easily refolded. Here, we present a method to incorporate synthetic peptides carrying multiple PTMs or ncAAs into both cytosolic and membrane proteins. The work is performed in live eukaryotic cells by tandem protein trans-splicing (PTS) using two orthogonal split intein pairs. We anticipate the approach to be broadly applicable as it allows for the introduction of a virtually limitless array of modifications, including those not accessible by existing methods.