•A RT-PCR based test and automated testing platform for MTB and drug resistance detection in a single run.•Includes all the reagents required to perform sample to result analysis in 2.5 h.•PCR components in lyophilized format.•Inclusion of controls for quality check, ensure result integrity and result reliability.•Analytical sensitivity of 25 CFU/ml for MTB detection, 200 CFU/ml for rpoB, inhA and katG genes.•Compatible with respiratory pulmonary and non- pulmonary specimens.•Storage and transport at ambient temperature (15–30 °C). A real time-polymerase chain reaction-based test in lyophilized form, was developed to simultaneously identify Mycobacterium tuberculosis complex (MTC) by targeting IS6110, rrs as dual markers, as well as mutations causing rifampicin and isoniazid resistance. The test was evaluated for pulmonary and non-pulmonary specimens from sample isolation to PCR analysis. The test demonstrated limit of detection of 25 CFU/mL for MTB, 200 CFU/mL for rpoB and inhA/katG targets with >95 % CI. The specificity for MTC was supported by a comprehensive clinical validation (n = 100). This load-and-go molecular platform, with features of high throughput, long shelf-life, room temperature storage provides simultaneous detection of MTC and its drug-resistant mutations in minimal time. The test named “PathoDetect TM MTB-RIF and INH resistance detection kit” has been approved by Central Drugs Standard Control Organisation, Indian Council of Medical Research and would have implications for tuberculosis elimination programs.