Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has been used to visualize molecular distributions in various biological samples. While it has succeeded in localizing molecules ranging from metabolites to peptides, quantitative MSI (qMSI) has remained challenging, especially in small biological samples like spheroids. Spheroids are a three-dimensional cellular model system that replicate the chemical microenvironments of tumors. This cellular model has played an important role in evaluating the penetration of drugs to better understand the efficacy of clinical chemotherapy. Therefore, we aim to optimize a method to quantify the distribution of therapeutics in a single spheroid using MALDI-MSI. Studies were performed with the therapeutic irinotecan (IR). The calibration curve showed a linear relationship with a limit of detection (LOD) of 0.058 ng/mm2 and R2 value at 0.9643. Spheroids treated with IR for different lengths of time were imaged using the optimized method to quantify the drug concentration during the penetration process. With a dosing concentration of 20.6 μM, the concentration of IR at 48 h of treatment was 16.90 μM within a single spheroid. Furthermore, spheroids were divided into different layers by spatial segmentation to be quantified separately. This MALDI-qMSI method is amenable to a wide range of drugs as well as their metabolites. The quantification results show great potential to extend this method to other small biological samples such as organoids for patient derived therapies.