Daphnia magna is a keystone indicator zooplankton used in environmental quality assessments. Comparative metabolomics, which contrasts small biomolecular regulations under different conditions, has emerged as a sensitive and informative investigation tool for the assessment of environmental stressors on D. magna. Baseline metabolomic variation is likely impacted by the asexual reproduction cycle of D. magna; however, the relationship between metabolite concentration regulation and reproduction cycle has not been investigated. This study investigated the time-course regulation of 51 metabolites during different reproduction stages to determine how the reproduction cycle controlled the metabolite profile of D. magna. Two-way analysis of variance (ANOVA) results reveal that most metabolites show significantly differentiated concentrations by individual or a combination of reproduction stages and sampling time. On the basis of the partial least-squares-discriminant analysis (PLS-DA) and ANOVA-simultaneous component analysis (ANOVA-SCA), stages 2 and 3 of reproduction show similarity in metabolite abundance regulation compared to stage 1. Metabolites were classified as being either dependent or independent of reproduction in the pairwise concentration correlation based on Pearson correlation coefficients. This study observed that the D. magna reproduction stage is an important consideration and potential variable and should be considered carefully when conducting metabolomic experiments using D. magna.