Mass spectrometry-based tissue profiling and imaging are technologies which allow identification and visualization of protein signals directly on thin sections cut from fresh frozen tissue specimens. These technologies were utilized to evaluate protein expression profiles in the normal mouse prostate during development (1 to 5 weeks of age), sexual maturation (6 weeks of age) and adult prostate (at 10, 15 or 40 weeks of age). The evolution of protein expression during normal prostate development and maturation were subsequently compared to 15 week prostate tumors derived from genetically engineered mice carrying the Large T antigen gene under regulation of the prostate-specific probasin promoter (LPB-Tag mouse model for prostate cancer). This approach identified proteins differentially expressed at specific time points during prostate development. Furthermore, expression of some of these proteins, for example probasin and spermine binding protein, were associated with prostate maturation, and prostate tumor formation resulted in their loss of expression. Cyclophilin A, a protein found in other cancers, was differentially a-acetylated on the N-terminal and both isoforms appeared during normal prostate and prostate tumor development. Imaging mass spectrometry localized the protein signals to specific prostatic lobes or regions. Thus, tissue profiling and imaging can be utilized to analyze the ontogeny of protein expression during prostate morphogenesis and tumorigenesis and identify proteins which could potentially serve as biomarkers for prostate cancer.