Background: The development of a highly effective whooping cough vaccine in the 1940s led to a decline in the number of cases. In the 1980s, researchers in Japan discovered an acellular vaccine. Nowdays, the number of cases of whooping cough in the world is increasing again. During 2023 and the first half of 2024, an increase in the number of whooping cough lab cases was registered in the Center for Microbiology at the Institute of Public Health of Serbia. Methods and Objectives: Serum or nasopharyngeal swabs of the patients were sampled either at the Batut Institute, or they were sampled at other health institutions from Belgrade and Serbia, with the exception of Vojvodina, and delivered in a transport container to the Batut Institute. DNA was extracted from each sample using the MagMAX Viral/Pathogen kit. The presence of B. pertussis was determined using two PCR assays, VIASURE Bordetella Real Time PCR Detection Kit and Bosphore B. pertussis/parapertussis Genotyping Kit, Anatolia Geneworks. Detection of IgG antibodies to Bordetella pertussis toxin was performed by ELISA method using tests manufactured by Euroimmun, PerkinElmer Germany Diagnostics GmbH. Results: Of the 1163 laboratory-detected positive results, 594 (51.1%) were detected serologically, while 569 (48.9%) were detected using the RT-PCR method. The largest number of samples was from the Belgrade region (928), followed by the South and Eastern Serbia regions (135) and the Sumadija and Western Serbia regions (77). The largest number of lab cases was in the age groups of 10-14 years (454), 15-19 years (224), over 20 years (217) and in children under one year of age (158). Conclusions: The reasons for the re-emergence may be: less effective long-term protection declining immunization coverage, asymptomatic transmission in adolescents and adults, genetic modifications of B. pertussis, increased awareness of the disease, and more readily available laboratory diagnostics.