AbstractWe describe a new method for phosphopeptide proteomics based on the solid‐phase synthesis of phosphopeptides on beads suitable for affinity pull‐down experiments. Peptide sequences containing the Bad Ser112 and Ser136 phosphorylation motifs were used as bait in affinity pull‐down experiments to determine their ability to bind 14‐3‐3 proteins. Support‐bound peptides were assembled directly on the solid support (PEGA) by standard solid‐phase synthesis through a BAL‐type handle. The peptides were varied in length and sequence. This synthetic strategy also allowed introduction of a soft electrophile (aldehyde) at the C terminus for potential activity‐based proteomics. The synthetic support‐bound Bad phosphopeptides were able to pull down 14‐3‐3ζ. Furthermore, Bad phosphopeptides bound endogenous 14‐3‐3 proteins, and all seven members of the 14‐3‐3 family were identified by mass spectrometry. In control experiments, none of the unphosphorylated Bad peptides bound transfected 14‐3‐3ζ or endogenous 14‐3‐3. We conclude that the combined synthesis and display of phosphopeptides on‐bead is a fast and efficient method for affinity pull‐down proteomics.