Perfluorooctane sulfonate (PFOS) is a persistent pollutant which is potentially harmful and bioaccumulative to aquatic organisms. To evaluate the regulatory alteration of select metabolites with PFOS exposure at early and typical acute exposure periods in an aquatic indicator species Daphnia magna, the hourly abundance of the twenty-three metabolites was investigated over 24 h. To evaluate the bioaccumulation potential of PFOS at a sub-lethal concentration in D. magna, the daily accumulation into D. magna for 16 days was also evaluated. Twenty-three targeted metabolites were quantified over 1 to 4 h and 21 to 24 h of PFOS exposure using liquid chromatography tandem mass spectrometry (LC-MS/MS). Daphnid to water PFOS concentration ratios were monitored separately over different days and life stages at 0 to 76 h and 2 to 16 days of PFOS exposure. The observed metabolite abundance and bioaccumulation in the exposed groups was compared between sampling times. The results reveal that sub-lethal PFOS exposure at 2 mg/L and 20 mg/L alters regulation of arginine, tyrosine and adenosine monophosphate which are directly and indirectly related to energy status. The temporal metabolic responses observed for the early exposure period (4 h), but not for the typical acute exposure period (24 h), suggest the dysregulation potency of PFOS on metabolite regulation of D. magna and the importance of early time-course monitoring approaches. Sixteen days of bioaccumulation monitoring showed that PFOS is more bioaccumulative in younger D. magna. The observation of time-dependent bioaccumulation of PFOS in D. magna requires further studies to define its precise mechanism. Interestingly, the bioaccumulation potential of PFOS was found to be consistent between 72 h and 16 day exposure periods. No difference on the body burden to water concentration ratio during about one third of the life span time (16 days), compared to the 72 h exposure, suggests that the prolonged exposure did not increase the bioaccumulation of PFOS in D. magna. This study demonstrates that the Daphnia metabolites are rapidly responding to sub-lethal PFOS exposure and provides information on life stage and time-dependent bioaccumulation potential of PFOS. As such, metabolite regulation is a sensitive indicator to sub-lethal PFOS exposure and can be informative when combined with other measures of toxicity.