Since tick-borne diseases (TBDs) incidence, both in human and animal populations, is increasing worldwide, there is the need to assess the presence, distribution and prevalence of tick-borne pathogens. Reliable estimates on tick-borne pathogens (TBPs) prevalence represent the public health foundation to create risk maps and take effective prevention and control actions against TBDs. Tick surveillance consists of collecting and testing (usually in pools) thousands of specimens. Construction and analysis of tick pools represent a challenge due to the complexity of tick-borne pathogens and tick-borne diseases ecology. The aim of this study is to provide a practical guideline on appropriate pooling strategies and statistical analysis of infection prevalence through: (i) reporting the different pooling strategies and statistical methodologies commonly used to calculate pathogen prevalence in tick populations and (ii) practical comparison between statistical methods utilising a real dataset of infection prevalence in ticks collected in Northern Italy. Reporting detailed information on tick pool composition and size is as important as the correct TBPs prevalence estimation. Among the prevalence indexes, we suggest using maximum-likelihood estimates of pooled prevalence instead of minimum infection rate or pool positivity rate given the merits of the method and availability of software.