We present here the synthesis of a highly O-carboxymethylated chitosan derivative. First, an improvedprotocol for the two-step synthesis of N-trimethyl chitosan (TMC) from chitosan was developed, yield-ing a maximum degree of quaternization (DQ) of up to 46.6%. Successively, the chitosan derivativeO-carboxymethyl-N-trimethyl chitosan (CMTMC) was synthesized from the TMC obtained by applyingan optimized synthesis pathway. In contrast to previous reports, the optimized protocol was shown toyield very high rates (>85%) of O-carboxymethylation of CMTMC, as shown by1H NMR and heteronu-clear single quantum correlation (1H–13C HSQC). Finally, in vitro cytocompatibility (viability >80%) of thepolymer was demonstrated using human fibroblasts.