Introduction: Venous serum and plasma are optimal specimens for serological testing but may be logistically infeasible. Dried blood spots (DBS) are a feasible alternative, provided results are adequately sensitive and specific. We aimed to assess the diagnostic accuracy of DBS to measure IgG and IgM antibodies for vaccine-preventable diseases and compare test validity of DBS with venous blood. Areas covered: In October 2020, we searched seven databases for peer-reviewed studies assessing the diagnostic accuracy of DBS specimens compared with serum in detecting antibodies to VPDs in humans. We extracted data and assessed risk of bias in all included studies. We calculated sensitivity and specificity with 95% confidence intervals for each index-reference test comparison. We narratively synthesized the identified evidence on diagnostic accuracy and blood collection and processing methods for DBS. Studies on measles and rubella IgG and IgM were the most frequently identified and reported generally high sensitivity and specificity. Expert opinion: Lack of standardization in collection, storage, and testing methods limited systematic comparison across studies. Our findings indicate a need for additional validation studies on the diagnostic accuracy of DBS to expand their use in serological surveillance. We recommend practical considerations to improve standardized reporting for DBS validation studies.