Thread spray ambient ionization uses single threads as a medium for sampling and ionization. This approach was demonstrated through the detection of various capsaicinoids from the interior of pepper fruits without destruction of the sample. Pepper residues present on the thread were analyzed by the application of DC voltage and solvent to cause field-induced charged droplet generation. Capsaicinoids extracted from the sample are contained in the electrosprayed droplets and transported to the mass spectrometer for characterization. The thread spray mass spectrometry method was optimized using commercially available materials like 100% cotton, cotton:polyester (35/65), 100% polyester, and nylon fabrics and subsequently applied for in-situ analysis of six different pepper fruits and pepper spray residues on fabrics. The results indicated that the special physico-chemical characteristics of threads allowed a rapid and convenient sampling and ionization of pepper products for analysis by mass spectrometry. The total capsaicinoid ion yields for the various pepper products correlated very well with that reported in Scoville Heat Units, suggesting that quantitative assessment of pungency levels may be achieved via the direct sample analysis without prior separation. Display omitted •Thread spray is capable of sustaining ion current longer than all other substrate-based ambient ionization techniques.•Thread spray ionization enables direct analysis of trace chemical residues on fabrics, without extensive sample preparation.•Thread is introduced as an effective way for storing forensic evidentiary garments, an approach expected to reduce backlogs.•Thread spray enables the sampling and quantitative analysis of fruit interior without complete destruction of the fruit.