The diagnosis of Hirschsprung disease (HD) depends on the histopathological analysis of rectal biopsies. This review aims to define the best rectal biopsy technique. A systematic literature review and proportional meta-analysis of the available case series studies of rectal biopsies were performed in this study. All case series with more than five rectal biopsies in children younger than 18 years of age suspected of HD that described at least one type of rectal biopsy were included. The studies that did not specify the rate of conclusive results and the rate of complications of the biopsy procedures were excluded. According to the literature review, there were four different techniques of rectal biopsy: open, suction, punch, and endoscopic. In the title and abstract screening process, we assessed 496 articles, 159 fulfilled the eligibility criteria, and 71 studies reported our outcomes of interest and were included in the meta-analysis. The pooled proportion of conclusive results was 94% in open biopsies (95% CI 0.89–0.98), 95% in punch (95% CI 0.90–0.98), and 88% in suction group (95% CI 0.85–0.92). The pooled proportion of complication rates was 2% in open biopsies (95% CI 0.00031–0.04), 0.039% in suction (95% CI 0.00023–0.0006), and 2% in punch biopsies (95% CI 0.00075–0.04). Suction, punch, and open techniques presented comparable rates of conclusive results. In the suction group, the association between different methods of histopathological analysis increased conclusive results rates; however, the punch biopsy was associated with significantly higher complication rates than the suction technique.