The steroid hormone 20-hydroxyecdysone (20E) controls molting in arthropods. The timing of 20E production, and subsequent developmental transitions, is regulated by a variety of factors including nutrition and photoperiod. Environmental factors, such as temperature, play a critical role in regulation as well. The increasing prevalence of urban heat islands (UHI), or areas with elevated temperature due to retained heat by built structures, in response to rapid urbanization has made it critical to understand how organisms respond to elevating global temperatures. Some arthropods, such as the Western black widow spider, Latrodectus hesperus, appear to thrive under UHI conditions, but the physiological mechanism underlying their success has not been explored. Recently, we have shown that L. hesperus, a troublesome urban pest, in fact responds to urban heat island conditions in Phoenix, AZ with delayed development, reduced body mass, and increased mortality. Here we look at the relationship between 20E levels and development in spiderlings reared under desert (27ᵒC), intermediate (30ᵒC), and urban (33ᵒC) temperatures, filling a noticeable gap in not only understanding ecdysteroids’ role in arachnid development but how incremental changes in environmental conditions affect the regulation of this process. Developmental progression and hemolymph 20E titers were recorded for several families of spiders collected from across the urban Phoenix area with data spanning from day 55 to 75 of development, focusing on the second developmental instar. We found that 33°C, but not 30°C, led to 1) a significantly higher production of 20E throughout development, 2) a reduced and delayed molt-inducing 20E peak, and 3) noticeable reductions in growth rate and mass. At 30°C, a variable response is seen in molt timing, without the negative impacts on size and mortality as seen at 33°C, suggesting that at UHI temperatures, the optimal developmental temperature threshold has been surpassed.