这阶段是确证选定靶标的调控会引发预期的生物效应并与临床治疗相关。常用的方法有基因突变、基因敲除、使用抗体、药物抗性突变和siRNA等。值得关注的是，51%的临床二期的失败都是由于药效不够，很多都归因于前期靶标确证工作的不足。这一阶段的失败随之而来的是后期巨额投入的付之东流。
疾病关联
临床靶标确证平台（https://www.targetvalidation.org/）整合了潜在药物靶标和疾病的关系，其中心思想是从多种类型数据发掘靶标和疾病的关联。DisGeNET（http://www.disgenet.org/web/DisGeNET/menu/home）是个整合了基因-疾病关系信息的药物发现平台，其数据来源是开源的数据库和文献。
化学探针
化学探针是针对某个靶标的已知小分子，能够行使预期的生物效应，但在某方面性质有所欠缺而不能成药。靶标确证时，选择高质量的探针非常重要，可以避免产生误导性结果。虽然化学探针可能被认为主要用来提供靶标效应和临床治疗效应的证据，但其对于解除靶标潜在的毒性风险也很重要。
值得注意的是，化学探针不是潜在的候选药物，其最关键的性质是对专利化合物及其代谢物的选择性。对化学探针而言，口服生物利用度的要求不是必须，了解探针的药代性质可以深入了解其PK/PD的关系。然而不是所有的化学探针都已被研究透彻，随着时间的推移，更多方面的信息会被发掘。因此有人开发了化学探针数据库，可供信息查询（http://www.chemicalprobes.org ）。
图1. 比对化学探针和药物的不同要求
基因敲除
基因敲除是一种遗传技术，通过使生物体的某个基因失效，对比正常组和敲除组的差异，从而推断出此基因的功能。然而，单个基因敲除可能对整个生物体的发育产生影响，因此已经开发出了敲除某特定组织中的基因或时间特异性敲除的技术。
图2. CRISPR-Cas9敲除介导的抗体特异性验证原理
小分子干扰RNA（siRNA）
siRNA是一类双链RNA，在RNA干扰的通路中发挥作用。siRNA作为互补核苷酸的序列，可以用来干扰特定基因的表达，从而有效地敲低目标基因。然而应该注意的是，不完全互补性的基因也可能被siRNA下调。
研究健康细胞中靶标的调节作用可以评估基于机制的毒性作用。由于多效性，相同的靶标可能在不同的器官系统中或在发育和成年期的不同时间点具有不同的功能。从敲除小鼠和人类的遗传缺陷中获得的数据可以进一步提供潜在不良反应的有关提示。
图3. siRNA的作用原理
Trim-Away
Trim-Away技术是另一种消耗内源性蛋白质的方法，在哺乳动物细胞中急剧降解内源蛋白质，而无需事先对基因组或mRNA进行修饰。 Trim-Away利用细胞蛋白质降解机制，仅需几分钟就可去除未修饰的天然蛋白质。关于此技术的更多信息可查阅 MRC website。
靶标确证研究的重要性
有些靶标确证实验的重现性受到质疑，具体数据很难估计，非官方报道此数字超过一半。这被认为是临床II期失败的主要原因，近三分之二的项目中，公布的数据与内部数据之间存在不一致，大大延长了靶标验证过程的时间，在大多数情况下导致项目终止（Arrowsmith J, Miller P. Trial watch: phase II andphase III attrition rates 2011-2012[J]. Nature Reviews Drug Discovery, 2013,12(8): 569-569.）。有几项研究着眼于分析重现性差的原因，如抗体的特异性差（Baker M. Blame it on the antibodies[J]. Nature,2015, 521(7552): 274.）、细胞系鉴定错误等。
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