To carry out this work, the researchers first developed the Micro-funnel from biocompatible fused silica capillary with a fine tip (~25 μm), enabling precise implantation into the spheroid to collect extracellular compounds. The spheroids, cultured using a colon carcinoma cell line (HCT-116), were treated with the anticancer drug irinotecan under various concentrations and durations. The Micro-funnel allowed for targeted sampling, isolating metabolites in a microscale environment that would otherwise be challenging to access without dilution or selection bias. Once metabolites were collected, the Single-probe was inserted into the opening of the Micro-funnel to extract these metabolites and analyzed by MS. The changes in the spheroid's extracellular lipid profile were observed, particularly in phospholipids and glycerides, with increased lipid abundance as treatment concentration and exposure time increased. These results indicated that irinotecan prompted significant shifts in lipid metabolites, which could contribute to drug resistance mechanisms within central tumor cells. This study's workflow demonstrates an effective methodology for profiling the extracellular environment of live spheroids, making it a valuable tool for investigating drug response, cellular communication, and resistance mechanisms in three-dimensional (3D) cancer models.