Opioid-like designer drugs represent one of the most rapidly evolving and analytically challenging subsets of new psychoactive substances (NPS). These compounds, which include fentanyl analogues, benzimidazole nitazenes, U-series opioids and other atypical μ-opioid receptor agonists, have repeatedly appeared on the illicit market in waves since the mid-2010s, often associated with clusters of fatal intoxications and with high potential for abuse. Their structural diversity and low active doses (often in the microgram range) create severe difficulties for detection and toxicological interpretation. This chapter provides a comprehensive overview of analytical issues encountered in the detection of opioid-like designer drugs, from screening to confirmation and quantitation. Emphasis is placed on the rapid turnover of emerging analogues, instability of the parent compounds, predominance of metabolites in biological matrices and the limited cross-reactivity of common immunoassays. Instrumental solutions, particularly LC–MS/MS, LC–HRMS, ion mobility and chiral workflows, are discussed together with pre-analytical aspects such as sample stability, matrix effects and microsampling strategies. Practical case examples and validated analytical procedures are drawn from recent literature, illustrating how multi-tiered and retrospective HRMS approaches can mitigate detection gaps and improve forensic and clinical response capabilities.