Passive acoustic monitoring (PAM) is increasingly being used for the survey of vocalising wildlife species that are otherwise cryptic and difficult to survey. Our study aimed to develop PAM guidelines for detecting the Yellow-bellied Glider, a highly vocal arboreal marsupial that occurs in native Eucalyptus forests in eastern and south-eastern Australia. To achieve this, we considered the influence of background noise, weather conditions, lunar illumination, time since sunset and season on the probability of detecting vocalisations. We deployed Autonomous Recording Units (ARUs) at 43 sites in the Central Highlands of Victoria during two periods: spring/summer (October 2018 to January 2019), and autumn/winter (May to August 2019). ARUs were programmed to record for 11 hours from sunset for 14 consecutive days during each period. Background noise resulted from inclement weather (wind and rain) and masked vocalisations in spectrograms of the recordings, thus having the greatest influence on detection probability. Vocalisations were most common in the four hours after sunset. Rainfall negatively influenced detection probability, especially during the autumn/winter sampling period. Detection of Yellow-bellied Gliders with PAM requires deploying ARUs programmed to record for four hours after sunset, for a minimum of six nights with minimal inclement weather (light or no wind or rain). The survey period should be extended to 12 nights when rain or wind are forecast. Because PAM is less labour intensive than active surveys (i.e., spotlighting and call playbacks with multiple observers and several nights’ survey per site), its use will facilitate broad-scale surveys for Yellow-bellied Gliders.