Streptococcus pneumoniae infections are often difficult to diagnose accurately, as it is not uncommon for clinical samples to be culture-negative, particularly after antibiotic administration. The rapid Binax NOW S. pneumoniae urinary antigen test lacks specificity in children, owing to pneumococcal antigen reactions in children who are nasopharyngeal carriers of S. pneumoniae. A western blot assay with a specific polyclonal antibody was developed for direct detection of the putative proteinase maturation protein A (PpmA) in urine samples from children with pneumococcal infections. The sensitivity and specificity of the assay were 66.7% and 100%, respectively. Previous antibiotic treatment or S. pneumoniae nasopharyngeal colonization did not affect PpmA antigenuria. Results also demonstrated the presence of PpmA cross-reactive epitopes in commensal bacteria that co-colonize the nasopharyngeal niche, although the non-pneumococcal cross-reactive protein(s) did not interfere with the detection assay. S. pneumoniae PpmA in the urine of children with pneumococcal infections may be a marker that has the potential to be used in the clinical diagnosis of pneumococcal infection.