Lung disease (LD) due to nontuberculous mycobacteria (NTM) becomes an important clinical concern, because the incidence and prevalence of NTM-LD have increased over the last decade. Among the NTM-LD in Taiwan, Mycobacterium avium complex (MAC) and M. abscessus (MAB) are the most frequent pathogenic species. Because MAC and MAB exist in the environment ubiquitously, clinical relevance of MAC isolated in sputum is only around 35~42%, and that of MAB is around 33%. According to the guideline of the American Thoracic Society, the diagnosis of MAC-LD and MAB-LD is based on clinical, radiographic, and mycobacteriologic criteria. For microbiology criteria, two or more sets of positive sputum mycobacterial culture for the same NTM species within one year is required. Actually, mycobacteria culture is neither timely nor efficient, which needs weeks to wait the results, especially for the slowly growing NTM. Moreover, the nucleic acid amplification method cannot discriminate true NTM infection and solely colonization because airway NTM colonization is not uncommon. Therefore, diagnosis of true NTM-LD is a big challenge in clinical practice. However, to early diagnose and then start treatment of NTM-LD is important because NTM-LD may be rapid lethal in critical status or in patients without early proper treatment. Hence, more rapid and accurate diagnostic test should be developed.