•Daptomycin initial dosage was adequate in 68.3% (43/63) of patients.•High variability in daptomycin use and serum levels was observed.•A trough serum level (Cmin) of <3.18 mg/L was independently related to poor outcome.•Therapeutic drug monitoring might be useful to optimise daptomycin doses and outcome. Therapeutic drug monitoring (TDM) could optimise daptomycin use. However, no validated serum target levels have been established. This prospective study at a tertiary centre including hospitalised patients receiving daptomycin aimed to evaluate the adequacy of daptomycin doses in a real-life study, assess interpatient variability in serum levels, identify predictive factors for non-adequate serum levels and assess their clinical impact. Blood samples trough (Cmin) and peak (Cmax) levels were drawn ≥3 days post-treatment initiation. Serum daptomycin concentrations were determined by HPLC. Outcome was classified as: (i) favourable, if clinical improvement or cure occurred with no adverse events; or (ii) poor, in the case of no clinical response, recurrence, related mortality or if adverse events were detected. Sixty-three patients (63.5% male; median age 63.0 years) were included. The most common indications for daptomycin use were bacteraemia (46.0%), complicated skin and soft-tissue infection (30.2%) and endovascular infection (15.9%). The initial dosage was adequate in 43 patients (68.3%), low in 14 (22.2%) and high in 6 (9.5%). Large interindividual variability in serum levels was observed, with a median Cmin of 10.6 mg/L (range 1.3–44.7 mg/L) and median Cmax of 44.0 mg/L (range 3.0–93.7 mg/L). Multivariate analysis showed that Cmin < 3.18 mg/L was independently related to poor outcome (OR = 6.465, 95% CI 1.032–40.087; P = 0.046). High variability in daptomycin use and serum levels was detected. Specific serum targets were identified as risk factors for poor outcome. TDM might be useful to optimise daptomycin doses and to avoid therapeutic failure.