The purpose of this study was to develop a Raman spectroscopy (RS) method as an effective tool for the non-intrusive pre-delivery analytical quality control (AQC) of two camptothecin analogs, i.e. irinotecan (IRI) and topotecan (TPT), which are prescribed and compounded at the hospital. Following a phase of analytical pre-validation, based on the actual conditions of use of the analogs, the protocol was validated and compared with the reference high-performance liquid chromatography (HPLC) method. For IRI, AQC by RS has been validated in ranges from 0.94 to 3.27 mg/ml in saline solutions and from 0.89 to 3.30 mg/ml in dextrose solutions. These ranges recover the entire therapeutic concentrations encountered in clinical practice, i.e. 1.08–2.8 mg/ml. The RS and HPLC methods were validated by calculating the accuracy profile and provided excellent results for the analytical validation key criteria. The Spearman and Kendall correlation tests (p-value < 1.10−11) and the statistical studies performed on the Bland and Altman graphs confirm a strong correlation between RS and HPLC results. However, we show that a routine apparatus is unable to quantify TPT therapeutic concentrations ranging between 25 and 50 µg/ml but that a sufficiently powerful RS bench is able to detect and quantify TPT. Overall, these results confirm the potential of the RS option for future innovative applications. Owing to its analytical and practical qualities, this promising method contributes to the improvement of the safety of the medication circuit at the hospital and to the protection of caregivers and their working environment. Copyright © 2015 John Wiley & Sons, Ltd.