The steroid hormone 20-hydroxyecdysone (20E) controls molting in arthropods. The timing of 20E production, and subsequent developmental transitions, is influenced by a variety of environmental factors including nutrition, photoperiod, and temperature, which is particularly relevant in the face of climate change. Environmental changes, combined with rapid urbanization, and the increasing prevalence of urban heat islands (UHI) have contributed to an overall decrease in biodiversity making 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. Here we examine the relationship between hemolymph 20E titers and spiderling development under non-urban desert (27°C), intermediate (30°C), and urban (33°C) temperatures. We found that a presumptive molt-inducing 20E peak observed in spiders at non-urban desert temperatures was reduced and delayed at higher temperatures. Intermolt 20E titers were also significantly altered in spiders reared under UHI temperatures. Despite the apparent success of black widows in urban environments, we noted that, coincident with the effects on 20E, there were numerous negative effects of elevated temperatures on spiderling development. The differential effects of temperature on pre-molt and intermolt 20E titers suggest distinct hormonal mechanisms underlying the physiological, developmental, and behavioral response to heat, allowing spiders to better cope with urban environments.