Effects of different matrices and primary ions on secondary ion emission efficiency were investigated for the analysis of D-tryptophan, C11H12N2O2, using secondary ion mass spectrometry (SIMS) under impact of 25-keV Bi+ and Bi3+. Matrix-embedded solid samples of D-tryptophan with alkaline earth metal salt of CaCO3 were prepared in the form of pellets with either additional cellulose or graphite. In both matrices, CaCO3/cellulose and CaCO3/graphite, the ionization of D-tryptophan primarily proceeds with a loss of small functional groups or by protonation, surprisingly no Ca cationization. Using Bi+, a decreased ion yield is observed with cellulose in comparison with graphite. Interestingly, using Bi3+ cluster-SIMS with the cellulose matrix shows enhancement of D-tryptophan molecular ions by a factor of 2, compared to the graphite matrix. The results of molecular ion yield improvement using graphite matrix support the importance of projectile type, either atomic or cluster. The differences might be explained by the matrix geometries, where cellulose forms a polymeric network compared to grains of graphite. Therefore, Bi+ is less effective due to single event impact, while Bi3+ is more effective due to larger mass and impact area within the cellulose polymeric network. Copyright © 2012 John Wiley & Sons, Ltd.