Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a well-known technique for polymer analysis, particularly for determining the molecular weight and structural details of dendrimers. In this study, we evaluated the performance of various matrices, such as 2′,4′,6′-trihydroxyacetophenone (THAP), α-cyano-4-hydroxycinnamic acid (HCCA), and sinapinic acid (SA), and their combinations, on the sensitivity and resolution of poly(amidoamine) (PAMAM) dendrimers of different generations (G3.0, G4.0, and G5.0). Our results demonstrated that the combination of HCCA-THAP significantly enhanced spectral resolution and peak intensity compared to individual matrices, particularly for higher-generation dendrimers. This improvement is attributed to the better ionization efficiency achieved with the combined matrices. These findings provide critical insights into optimizing MALDI-TOF MS for the accurate characterization of complex polymers, with potential applications in drug delivery and nanotechnology.