Accurate estimation of invertebrate biomass is essential for studies of ecosystem structure and function. To estimate seasonal changes in spider biomass on canopies of two coniferous and three broadleaf tree species in plantation and native forests of subtropical Taiwan, 6,554 spiders were collected from February to August 2015. A part of the collection (n = 887) was weighed and measured to develop appropriate body size-mass equations for subtropical montane forest systems. By comparing different equations, we demonstrate that use of shape-specific equations and incorporation of body width measurements provide the most accurate biomass estimates. Generalized whole-fauna equations produced accurate estimates for the entire pooled sample but caused relatively higher biases than shape-specific equations when applied to spider assemblage subgroups. Applying coefficients derived from the best equations, we describe the seasonal variation in spider biomass on five dominant tree species. Compared to broadleaf trees, Taiwan cedar and Japanese cedar supported higher spider biomass and abundance. Spider biomass on the two conifers declined in early spring, then increased over time, and reached its highest in August. In contrast, spider biomass on caudate-leaved chinkapin and Mori oak formed bell-shaped curves with a high peak in late spring. Spider biomass on deciduous Formosan alder remained at a low level from February to June and increased in August. Moreover, phenological patterns of spider biomass differed from those of abundance on some tree species. We expect that regression parameters from our study are appropriate for biomass estimation of spider assemblage in subtropical montane forest systems.