Protothecosis is a severe form of mastitis in dairy cows caused by colorless algae of the genus Prototheca. Since P. zopfii is highly resistant to all known chemotherapeutics, infected cows must be removed from the herd. Eradication measures are difficult since many chronically infected cows may become intermittent shedders. Therefore, cultural methods are insufficient for control measures. In order to eradicate Prototheca zopfii-mastitis in dairy cattle herds, two isotype specific indirect ELISA for detection of IgA and IgG1 in whey were used in a dairy herd highly affected with protothecal mastitis. All cows (n = 313) were tested four times in intervals of six months. Milk specimens were examined in parallel by cultivation and serologically using two indirect ELISA systems for specific IgA and IgG1 in whey. Cows tested Prototheca positive were consequently separated from the herd and slaughtered. At the first examination, 15.6% of the animals were found positive by culture, and 23.3% were positive in at least one of the ELISA systems. Within two years, protothecal prevalence and incidence decreased to zero indicating that the eradication strategy used was successful. In summary, serological identification of P. zopfii-infected lactating cows is an useful tool to eradicate protothecal bovine mastitis in infected herds.