Gut content analysis of common predators such as spiders using DNA metabarcoding has facilitated the understanding of community level predator-prey relationships in ecosystems. To interpret the DNA metabarcoding result correctly knowledge of different factors influencing persistence of prey DNA in the predator gut is crucial. Our study investigated four such factors; i.e., the effects of (1) starvation time prior to feeding; (2) time elapsed since feeding; (3) spider size; and (4) presence of egg sacs or spiderlings on the detection rate of prey DNA in the gut contents of field-collected Lycosidae spiders. We performed feeding trials in a controlled lab environment and analyzed predator gut content data using PCR and DNA metabarcoding techniques. We used two primer combinations (NoSpi2 with BR2 or fwhR2n) and three different prey species Drosophila hydei, Gryllus bimaculatus, and Coecobrya tenebricosa in our feeding trials. The NoSpi2-fwhR2n primer combination was more effective at amplifying a PCR product of the correct size compared to the NoSpi2-BR2 combination and was therefore used for the DNA metabarcoding. The results showed that time since feeding significantly influenced the detection rate of Drosophila and Gryllus DNA, with longer time intervals leading to decreased detection odds. Spider size, starvation time, and the presence of egg sacs or spiderlings had minimal or no influence on prey detection. This study highlights the importance of considering different factors that influence prey DNA detection in spider diet studies and using appropriate primers. Future research should account for variables such as prior gut contents, starvation period, and their potential interaction with spider metabolic rate.