The stability of antimicrobials in peritonitis during peritoneal dialysis (PD) solutions is a critical factor influencing treatment success. This study investigated the stability of daptomycin (DAP) when combined with icodextrin-based PD solution, by measuring DAP concentrations and observing any structural changes. A dose of DAP (350 mg) was dissolved in 7 mL of saline in a clean bench. The solution was then injected into the large compartment (1,260 mL) of NICOPELIQ® Peritoneal Dialysis Solution and thoroughly mixed. Samples were collected at intervals ranging from 0 to 336 h (7 points in total). The concentration of DAP was quantified using high-performance liquid chromatography (HPLC). The structure of any unidentified peaks was determined using HPLC coupled with electrospray ionization tandem mass spectrometry. DAP maintained 90% of the initial concentration in NICOPELIC® for 72 h at room temperature and 12 h at 37 °C. Unidentified peaks, distinct from DAP, were detected during analysis. Further investigation indicated that these peaks corresponded to anhydrated DAP. The findings from this stability study are expected to enhance the effectiveness of outpatient management and preparation for treating peritonitis using DAP.