Kingella kingae is currently recognized as the prime etiology of skeletal system infections in children aged 6-48 months. The organism is notoriously fastidious, its growth is inhibited by synovial fluid and bone exudates, and its presence in clinical specimens is commonly missed by traditional culture methods. Areas covered: The present review discusses the use of improved laboratory methods to detect the organism in normally sterile body fluids, exudates, and upper respiratory tract specimens. Expert commentary: While inoculation of joint and bone exudates into blood culture vials dilutes the concentration of detrimental factors and significantly improves the isolation of the organism, novel PCR-based assays have enhanced sensitivity, shortened the time-to-detection of K. kingae from 3-4 days to <24 h, and enabled the bacteriological diagnosis in patients being administered antibiotic therapy. PCR-based assays that amplify the 16S rRNA gene results in a 200% improvement in the diagnosis of the organism compared to culture, whereas the use of real-time PCR tests that target K. kingae-specific DNA sequences increases the detection rate by a five-fold factor and reduces the fraction of culture-negative septic arthritis and osteomyelitis in infants and young children.