Quantifying the growth of entomopathogenic fungi is crucial for understanding their
virulence and pathogenic potential. Traditional methods for determining growth, such as biomass
determination or colony growth area, are time-consuming and quantitatively and spatially limited in
scope. In this study, we introduce a high-throughput method for rapidly measuring fungal growth
using spectrophotometry in small-volume, liquid media cultures in 96-well microplates. Optical
density (OD) changes were directly correlated with dry weight of samples for six isolates from
three species of the genus Metarhizium to validate spectrophotometric growth measurements, and
investigate species- and isolate-specific effects. We quantified fungal biomass from the microcultures
by extracting, drying, and weighing mycelial mats. From the relationship established between OD
and biomass, we generated standard curves for predicting biomass based on the OD values. The
OD measurements clearly distinguished growth patterns among six isolates from three Metarhizium
species. The logistic growth phase, as captured by the OD measurements, could be accurately
assessed within a span of 80 h. Using isolates of M. acridum, M. brunneum, and M. guizhouense,
this technique was demonstrated to be an effective, reproducible, and simple method for rapidly
measuring filamentous fungal growth with high precision. This technique offers a valuable tool for
studying the growth dynamics of entomopathogenic fungi and investigating the factors that influence
their growth.