© 2022 Elsevier B.V.Antibiotic resistance is one of the most pressing scientific and societal issues of our age. There is an urgent need to develop new diagnostic technologies which can quickly determine whether an infection is susceptible or resistant to different treatments so that rational antibiotic prescribing can take place. The main objective of the study was therefore to develop a rapid, simple, cost effective and comprehensive antibiotic susceptibility/resistance test based on rapid nucleic acid profiling. To do so, we integrated a microelectrode sensor within a microfluidic chip that combined bacterial incubation, lysis, and electrochemical detection chambers in a single simple set-up. As a case study, Mycobacterium smegmatis was investigated as a surrogate organism for Mycobacterium tuberculosis. The novelty of the work lies in developed capability of performing incubation, lysis, fragmentation, and detection process in a comprehensive yet simple lab-on-a-chip device called ‘MycoCHIP’. A gold microelectrode in combination with a specifically developed nucleic acid probe sequence for the 16SrRNA region of the mycobacterial genome were employed to monitor M. smegmatis nucleic acid sequences using Differential Pulse Voltammetry (DPV) and Square-Wave Voltammetry (SWV). The results demonstrated that it was possible to detect bacterial nucleic acid sequences and distinguish antibiotic incubated (Ab-i) cells from nonincubated (Ab-n) cells on MycoCHIP with a label-free molecular detection. The antibiotic susceptibility test showed that through measuring 16SrRNA levels from M. smegmatis,sensitivity to antibiotic was apparent after 24 h incubation, with a developed protocol representing a potential approach to determining antibiotic susceptibility more quickly, reliable and economically than current methods.