Toxoplasma gondii is an obligate intracellular parasite that can cause severe disease in immunocompromised individuals and congenitally infected neonates. In order to determine whether serum peptide profile would reveal disease markers or allow determination of toxoplasmosis aggressiveness, mouse sera were collected from acutely infected, chronically infected and control subjects, and analyzed by using a quantitative label-free pepdomics approach (LC-MS/MS). Six hundred and seven endogenous peptides were identified among all the samples, with peptide profiling of difference that readily distinguished between acutely infected samples and other samples. Among these peptides detected in this study, 81 and 68 differentially expressed peptides (DEPs) were found in the acute and chronic infection stages, respectively. Through Gene Ontology analysis, most of the precursor proteins of these DEPs were associated with biological regulation and binding activity. The findings in this study will help in the search of peptide targets with a key role in disease diagnosis and create new opportunities for the development of better means for toxoplasmosis prevention.