Sepsis continues to pose a major challenge to global public health, with a high mortality rate. The current diagnostic tools to determine and initiate the medication are time-consuming and require intensive lab procedures. Q-linea AB launched a novel diagnostic instrument under the name ASTar® intending to provide minimum inhibitory concentration (MIC) values within six hours in a fully automated manner. The ASTar disc is the part of the instrument that contains the drugs formulated to be soluble during the experiment and stable over the shelf-life of the disc. In this study, formulations of five gram-negative antibiotics were developed and optimized to be included in ASTar discs. It is also to study their performance in the ASTar and their stability during storage. The formulations were developed by screening various excipients and solvents using high-performance liquid chromatography (HPLC) and broth microdilution tests (BMD). The chemical stability of the drugs loaded in the ASTar disc were investigated using liquid chromatography-mass spectrometry (LC-MS) after production (time zero) and after two months of storage at 25°C and 40°C. The concentration of excipients used in the formulation confirmed that it had no impact on bacterial growth by BMD analysis. The discs were produced successfully, as confirmed by the disc results by ASTar, where the drugs achieved the corresponding MIC values. There were no statistically significant differences in the concentration of the drugs observed between the discs analyzed at zero time and after two months at 25°C. The concentrations of drugs stored at 40°C were statistically different from that at zero time points, indicating some degradation. Using the formulation strategy applied in the study, it was possible to achieve correct MIC using even non-iron-depleted Mueller Hinton Broth (MHB). The study shows that the discs produced utilizing the developed and optimized formulas have the potential to be employed with the ASTar instrument. The stability study must be followed for an extended period to determine the performance of different formulations over time.