In this paper, the comparison of two automated high-performance liquid chromatography (HPLC) screening systems, a newly developed toxicological identification system (TOX.I.S.) versus the commercially available Remedi-HS (Bio-Rad), is presented. Urine samples from 405 cases screened positive for amphetamines, cocaine, and opiates by immunological assays and confirmed by GC-MS were analyzed with both systems. In more than 80% (TOX.I.S.) and 78% (Remedi-HS) of the cases (except for cocaine), the results obtained by both HPLC methods showed agreement with the earlier obtained results by immunoassay prescreening and gas chromatography-mass spectrometry (GC-MS). The evaluation showed that both automated HPLC methods led to comparable results and can be used alternatively. As the confirmation results for cocaine were rather poor (45% TOX.I.S., 54% Remedi-HS) in comparison to GC-MS, the TOX.I.S. was further optimized for the detection of the cocaine metabolite benzoylecgonine (BEC). The BEC method improved the detectability of BEC from 45% to 80%. Besides confirmation screening, the use of both systems in cases of acute intoxications was investigated. Information about basic compounds was obtained from urine screening by both systems, which therefore were useful as complementary techniques in the toxicological laboratory. The TOX.I.S. offers advantages such as common equipment, modern software, and higher versatility with the opportunity to establish additional methods in the system.