Acid mine drainage (AMD) is one of the main environmental problems in mining operations. The objective of this study was to assess AMD obtained from a copper mine via toxicity identification evaluation (TIE) using Daphnia magna and Chlorella vulgaris as indicator organisms. AMD was fractionated via filtration and aeration at pH 3 and 11, activated carbon, cation resin, anion resin, and ethylenediaminetetraacetic acid (EDTA). The results showed that unfractionated AMD has a low organic matter content (total chemical organic demand, CODT-183.05 mg/L), low pH (3.9), and high sulfates concentrations (2900 mg/L) and metal ions in solution (0.2-418.9 mg/L), producing high toxicity to Daphnia magna (0.00016% v/v) and no observable acute toxicity to Chlorella vulgaris (72 h-RFU 64.9%). For Daphnia magna, TIE fractionations with the greatest reduction in acute toxicity (LC50) were filtration/pH11 (non-toxic) and anion resin (LC50 = 0.43% v/v), with toxicity reduction percentages of 100% and 99%, respectively. Because of this, Cu was determined to be the main cause of acute toxicity to Daphnia magna. For Chlorella vulgaris, the activated carbon fraction stands out, increasing the % relative fluorescence units by 4% from 48 h to 72 h, demonstrating tolerance to AMD. The TIE technique is presented as an effective strategy to identify toxic compounds in complex samples and evaluate their effect on environmentally relevant organisms. Therefore, this study allows the analysis of the ecological risk in aquatic environments affected by mining activities, which supports environmental decision-making and the design of efficient treatment strategies.