An on-chip pulse-heating ionization source for protein samples was developed for the realization of miniaturized mass spectrometry. A protein analyte was ionized on a chip by applying only thermal energy to the solid phase sample without a laser, high voltage, or heated ambient gases. A fabricated ionization source consisting of a Pt/Cr microheater (width: 30 μm; length: 100 μm) on a silicon substrate was coupled with a time-of-flight mass filter to analyze a protein sample of bovine serum albumin (BSA, M = 66 kDa). A singly charged BSA ion and other multiply charged BSA ions were generated in the presence of 2,5-dihydroxybenzoic acid as a matrix. To detect the singly charged BSA ion, the required surface energy density of 1.65 × 10(-2) μJ/μm(2) was applied to the microheater for 500 ns. The use of the 2,5-dihydroxyacetophenone matrix resulted in the generation of the multiply charged protein analyte, while the use of the sinapic acid matrix showed abundant peaks in the low m/z region.