Awareness of human rights and related principles, such as equality and non-discrimination, is growing in sports. While debates on doping regulation typically target the contours of the prohibition and the sanctioning regime, much less attention has been given to how anti-doping detection impacts the level playing field, i.e. whether equality is realised in the manner in which the substances and methods are detected in athletes’ samples, or whether athletes are all equal when it comes to the analytical cut-offs that the regulations set. This article seeks to fill this gap and explores the implications of differentiation—or non-differentiation—in anti-doping detection for principles of equality and non-discrimination. After discussing notions related to equality in anti-doping detection, the article presents case studies from current anti-doping analytics, to make differentiation in that context tangible. Based on case law of the European Court of Human Rights, the Swiss Supreme Court and the Court of Arbitration for Sport, we submit that anti-doping authorities should resort to an operational ‘discrimination test’ when drafting technical regulation for anti-doping, in order to incorporate these principles ‘by design’ into the detection system. The article also demonstrates that—apparently—technical rules are not value-neutral, but that scientific data and policy choices are entwined in a way that warrants debate on the political scene, and creates duties of transparency and justification on part of the decision-makers.