Antibody against common carbohydrate moieties of macrolide antibiotics were generated in result of rabbit immunization with BSA-clarithromycin (CLA). For fabrication of immunochromatographic strip test, blue latex particles were labeled with antibody; GEL-CLA and anti-rabbit IgG were spotted on nitrocellulose membrane in test and control zones for the rapid simultaneous group determination of six macrolide antibiotics. Through careful optimization of parameters, namely the type of nitrocellulose membrane, the carrier for the coating antigen, its loading onto the strip (the number of nanodrops per spot, the number of spots per line and the number of lines), the loading of antibodies on the latex bead and the volume of the latex bead and stabilizing solutions, the color intensity was improved 100 times. CLA, roxithromycin, erythromycin, dirithromycin, azithromycin, and oleandomycin in buffer were visually detected at 1-1000 ng/mL level. The limits of instrumental detection were respectively 0.12, 0.15, 1.4, 2.1, 2.4, and 3.3 ng/mL. The composition of breast milk changes significantly both during a breastfeeding and during lactation period: protein levels decrease slightly, while carbohydrates increase, the fat content almost doubles. Therefore, for the unification of sample composition and avoiding matrix effect, a special 20-min procedure with methanol/hexane extraction was developed. Thus, the developed rapid on-site diagnostic assay format is suitable for monitoring of antibiotic content in breast milk during medication with any of six mentioned macrolides to ensure safe breastfeeding of infants.