An efficient procedure to access tritium-labeled maleimide derivatives in a high specific activity has been developed. N-Substituted maleimides containing the hydroxy functionality are O-methylated in a three-step synthesis route, including (1) Diels–Alder protection of the maleimide core, (2) O-methylation by the use of commercially available [3H]methyl nosylate, and (3) deprotection by retro-Diels–Alder reaction. With our procedure, N-hydroxyalkyl maleimide derivatives can be labeled in overall radiochemical yields of 13–15% and in >98% radiochemical purity. The major advantage of N-alkoxy maleimides in comparison to N-alkylated maleimides such as N-ethylmaleimide is their lower volatility, which enables safer handling with respect to radiation-safety protection. Tritium-labeled maleimide building blocks allow subsequent Michael-type conjugation reactions of thiol-containing biomolecules for mechanistic in vitro or in vivo studies.