Purpose: DNA is a highly valuable lead to identify people who were possibly involved in a crime. Even by small contact events, minute amounts of DNA (‘trace DNA’) can be transferred from a DNA source to an evidentiary item, which can be enough for a successful DNA analysis. The focus of this research is to get more insight in the collection of trace DNA from textiles by ‘stubbing’, which is a tape-lifting method using double-sided tape placed on a stub. The relation between the ‘stubbing force’ (the normal force that is applied during stubbing) and the collection efficiency of microspheres is investigated. Methods: Microspheres (Ø25 μm) were used as mock traces to mimic DNA-containing micro-traces. The particles were applied to textile substrates in a suspension of ethanol that was left to evaporate before sampling. Experiments were performed on three different polyester substrates. Traces were collected by stubbing while using 5 different stubbing forces. The number of microspheres placed on each substrate was counted before sampling and all stub-tapes were analysed after sampling to count how many of the microspheres were picked up, both by using stitched images from a digital light microscope. Custom-made image recognition software was used to automatically count the microspheres. Results: On all tested polyester substrates, the mean efficiency of the collection of microspheres increased with increasing stubbing force in a concave down increasing function. The increase of collection efficiency stagnated around 3–12 N, depending on the substrate material. The theoretical maximum collection efficiencies varied between 38% and 78%, depending on substrate material as well. Conclusions: Stubbing with a force higher than 12 N does not notably influence the collection efficiency from the variety of textiles that were tested. However, because the theoretical maxima of the collection efficiencies were far from 100%, it is highly likely that stubbing multiple times on the same spot of a substrate increases the total collection efficiency. The gained knowledge will help to standardize and improve the effectiveness of stubbing.