Folic acid (FA)-induced acute kidney injury (FA-AKI) is an increasingly prevalent rodent disease model involving the injection of a high dose of FA that culminates in renal FA crystal deposition and injury. However, the literature characterizing the FA-AKI model is sparse and dated in part due to the absence of a well-described methodology for the visualization and quantification of renal FA crystals. Using widely available materials and tools, we developed a straightforward and crystal-preserving histological protocol that can be coupled with automated imaging for renal FA crystal visualization and generated an automated macro for downstream crystal content quantification. The applicability of the method was demonstrated by characterizing the model in male and female C57BL6/JRj mice after 3 and 30 h of FA treatment. Kidneys from both sexes and timepoints showed a bimodal distribution of FA crystal deposition in the cortical and medullary regions while, compared with males, females exhibited higher renal FA crystal content at the 30-h timepoint accompanied by greater kidney weight and higher plasma urea. Despite comparable plasma phosphate concentrations, FA-AKI resulted in a substantially more elevated plasma intact fibroblast growth factor 23 (FGF23) in females, reflected by a similar pattern in osseous mRNA expression. Therefore, the presented method constitutes a valuable tool for the quantification of renal FA crystals, which can aid the mechanistic characterization of the FA-AKI model and serves as a means to control for confounding changes in FA crystallization when using the model for investigating early and prophylactic AKI therapeutic interventions. Here, we describe a novel method for the visualization and quantification of renal folic acid (FA) crystals in the rodent FA-induced acute kidney injury (FA-AKI) model. The protocol involves a straightforward histological approach followed by fully automated imaging and quantification steps. Applicability was confirmed by showing that the FA-AKI model is sex-dependent. The method can serve as a tool to aid in characterizing FA-AKI and to control for studies investigating prophylactic therapeutic avenues using FA-AKI.