The claims describe a process for designing and validating peptide-based substrates for a specified kinase. The process uses a rational approach to design peptides with a specific amino acid that is phosphorylated by a target tyrosine kinase, and upon phosphorylation shows an increased affinity for a lanthanide ion that results in an enhanced fluorescent signal. The process uses kinase specific input data sets, encodes the input into positional scoring matrices, analyses the positional scoring matrices in order to generate a candidate list of peptide sequences, compares the peptide sequences in the candidate list, substitutes residues at non-selective positions in selections from the candidate list with terbium binding residues, and then screens the substituted selections with different kinases in vitro.