Western blot analysis is widely used for detecting protein expression, analysis of protein–protein interactions, and searching for new biomarkers. Also, it is a diagnostic tool used for detection of human diseases and microorganism infections. Some Streptococcus pneumoniae proteins are important virulence factors and a few of them are diagnostic markers. Here, we describe the detection of two pneumococcal proteins, pneumolysin and PpmA, in human urine by using monoclonal and polyclonal antibodies.