Objective To evaluate culture‐independent procedures (immunochromatography and quantitative polymerase chain reaction [qPCR]) in the detection and susceptibility of Streptococcus pneumoniae directly from culture‐negative pleural fluid (PF) in children. Method Detection of S. pneumoniae in PF of children with parapneumonic effusion and/or empyema by using two culture‐independent methods: an immunochromatographic membrane test (IMT) which identifies the pneumococcal C antigen, and a real‐time PCR test to detect pneumococcal genes lytA and pbp2b, a marker of susceptibility of β‐lactam agents, in PF samples. Results We tested 36 PF specimens and recorded the previous use of antimicrobials. In the final analysis, 34 samples were included. IMT and qPCR presented positive results in 23 (67.6%) and 24 (70.6%) of the samples, respectively, showing a moderate agreement (k = 0.518) between the two methods. From the 36 children included, 34 (94.4%) had antibiotic data available by the time when PFs were collected. Thirty‐four (100%) children had been given treatment before PF sampling, with 33 (97%) receiving β‐lactam antibiotics administered empirically. Of the 24 lytA real‐time positive samples, 21 (87.5%) were also positive for pbp2b, a marker of β‐lactam susceptibility. Conclusion The reduced sensitivity of culture for pneumococcal detection can be improved through the addition of IMT and qPCR analysis. The utility of qPCR combining detection of lytA and a marker of β‐lactam susceptibility should be explored further.