〔Abstract〕 Objective To explore the direct-on-target micro-droplet growth assay(DOT-MGA) for rapid detection of the susceptibility of tigecycline and polymyxin. Methods A total of 67 strains ofKlebsiella pneumoniaewere collected for DOT-MGA.6 μl droplets with or without tigecycline or polymyxin(The final drug concentration is 2 μg/ml) were added in triplicate into the wells of the MALDI plate with four incubation time points(3 h, 4 h, 6 h, and 8 h).The results were classified as susceptible(score<1.7) and non-susceptible(score≥1.7) according to the Bruker Biotype software. Results After incubation for 4 h, the growth efficiency, specificity and positive predictive value of tigecycline and polymyxin were both 100.00%.The classification consistency rate was 98.15% and 96.15%,the sensitivity was 96.30% and 92.31%,and the negative predictive value was 96.45% and 92.86%,respectively. Conclusion DOT-MGA can provide rapid and reliable drug susceptibility diagnosis of tigecycline and polymyxin, which is of great significance for the clinical anti-infective treatment.