Two commonly used methods for cyanotoxin analysis are enzyme‐linked immunosorbent assay (ELISA) and liquid chromatography/tandem mass spectrometry (LC–MS/MS). Two rounds of interlaboratory comparisons of ELISA and LC–MS/MS analyses were conducted with 12 participating laboratories to evaluate method performances in various matrices, including cyanobacterial bloom and drinking water samples. Fifteen cyanotoxins, including 12 microcystin variants, nodularin, anatoxin‐a, and cylindrospermopsin were evaluated. The impact of sample matrices, preservatives, and quenching reagents was assessed, and no substantial effects were observed. Overall, comparable results were obtained among laboratories performing ELISA and LC–MS/MS analyses, respectively. ELISA results for fortified samples matched more closely with those from LC–MS/MS when microcystin cross‐reactivities were considered, providing data 26% closer to theoretical values on average. This study demonstrates that understanding the effect of cross‐reactivities when comparing ELISA and LC–MS/MS results and considering potential variabilities in commercial standards is important when interpreting data from these two methods.