We have applied protein expression profiling technologies in combination with immunohistochemistry, using fresh tissue and urine samples, to assess bladder cancer heterogeneity and prognosis as well as to generate protein markers for tumor progression and early diagnosis of the disease. Here, we review some selected lines of investigation and approaches undertaken by our laboratory, drawing on more than 15 years of experience in bladder cancer pboteomics, to high-light a number of issues that may be useful for researchers entering the field. In particular, we address the identification of markers for bladder cancer progression and exemplify the potential of gel-based proteomic profiling of urine samples for the early detection of urothelial carcinomas. In addition, we provide a brief description of a novel and highly promising source of biomarkers, the tumor interstitial fluid (TIF) that perfuses the tumor microenvironment.