This protocol describes a method to extract ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) from diatoms (Bacillariophyta) to determine catalytic performance. This protocol has been adapted from use in cyanobacteria and higher plants (Andrews, 1988; Whitney and Sharwood, 2007). First part (steps A1-A3) of the extraction provides a crude extract of Rubisco that is sufficient for carboxylation assays to measure the Michaelis constant for CO₂ (K_C) and the catalytic turnover rate (k_cat^c). However, the further purification steps outlined (steps B1-B4) are needed for measurements of Rubisco CO₂/O₂ Specificity (S_C/O, [Kane et al., 1994]).