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Abstract Phytoplankton biomass is routinely estimated using relationships between cell volume and carbon (C) and nitrogen (N) content that have been defined using diverse plankton that span orders of magnitude in size. Notably, volume has traditionally been estimated with geometric approximations of cell shape using cell dimensions from planar two-dimensional (2D) images, which requires assumptions about the third, depth dimension. Given advances in image processing, we examined how cell volumes determined from three-dimensional (3D), confocal images affected established relationships between phytoplankton cell volume and C and N content. Additionally, we determined that growth conditions could result in 30–40% variation in cellular N and C. 3D phytoplankton cell volume measurements were on average 15% greater than the geometric approximations from 2D images. Volume method variation was minimal compared to both intraspecific variation in volumes (~30%) and the 50-fold variation in elemental density among species. Consequently, C:vol and N:vol relationships were unaltered by volume measurement method and growth environment. Recent advances in instrumentation, including those for at sea and autonomous applications can be used to estimate plankton biomass directly. Going forward, we recommend instrumentation that permits species identification alongside size and shape characteristics for plankton biomass estimates.