ABSTRACT Biofilm represents a major public health concern. It is a highly structured and heterogeneous microbial population that is well protected by a hydrated extracellular matrix. In most cases, the difficulties in combating a wide spectrum of biofilm-associated diseases are due to the presence of dormant cells and differential molecular expression. Proteomics is the large-scale and systematic study of cellular proteome expression at any given time by mass spectrometry. It allows high-sensitivity and high-specificity identification of differentially expressed proteins in the biofilms. Over the past few decades, multiple lines of proteomic works have successfully elucidated various aspects of the biofilm including developmental stages, antimicrobial resistance, and survival mechanisms. However, the heterogeneity of biofilms may contribute to inconsistent proteome expression throughout a proteomic experiment. This is due to the fact that the mature biofilm is often associated with the mixture between monolayer and multilayer biofilms, thick microbial population, and chemical gradient of nutrients. This review highlights the biofilm heterogeneities, the principle of mass spectrometry in proteomics, and the possible strategies for quantitative proteomic analysis of heterogeneous biofilms. It is suggested that isolation of monolayer biofilm, laser capture microdissection, flow cytometry, and subtractive proteome profiling may be considered for an accurate and reliable quantitative proteomics experiment.