Background and purpose Opioids are potent analgesics. Opioids exert effects after interaction with opioid receptors. Opioid receptors are present in the peripheral- and central nervous system (CNS), but the analgesic effects are primarily mediated via receptors in the CNS. Objective methods for assessment of opioid effects may increase knowledge on the CNS processes responsible for analgesia. The aim of this review was to provide an overview of the most common objective methods for assessment of the spinal and supraspinal effects of opioids and discuss their advantages and limitations. Method The literature search was conducted in Pub Med (http://www.ncbi.nlm.nih.gov/pubmed) from November 2014 to June 2016, using free-text terms: “opioid”, “morphine” and “oxycodone” combined with the terms “pupillometry,” “magnetic resonance spectroscopy,” “fMRI,” “BOLD,” “PET,” “pharmaco-EEG”, “electroencephalogram”, “EEG,” “evoked potentials,” and “nociceptive reflex”. Only original articles published in English were included. Results For assessment of opioid effects at the supraspinal level, the following methods are evaluated: pupillometry, proton magnetic resonance spectroscopy, functional resonance magnetic imaging (fMRI), positron emission tomography (PET), spontaneous electroencephalogram (EEG) and evoked potentials (EPs). Pupillometry is a non-invasive tool used in research as well as in the clinical setting. Proton magnetic resonance spectroscopy has been used for the last decades and it is a non-invasive technique for measurement of in vivo brain metabolite concentrations. fMRI has been a widely used non-invasive method to estimate brain activity, where typically from the blood oxygen level-dependent (BOLD) signal. PET is a nuclear imaging technique based on tracing radio labeled molecules injected into the blood, where receptor distribution, density and activity in the brain can be visualized. Spontaneous EEG is typically quantified in frequency bands, power spectrum and spectral edge frequency. EPs are brain responses (assessed by EEG) to a predefined number of short phasic stimuli. EPs are quantified by their peak latencies and amplitudes, power spectrum, scalp topographies and brain source localization. For assessment of opioid effects at the spinal level, the following methods are evaluated: the nociceptive withdrawal reflex (NWR) and spinal EPs. The nociceptive withdrawal reflex can be recorded from all limbs, but it is standard to record the electromyography signal at the biceps femoris muscle after stimulation of the ipsilateral sural nerve; EPs can be recorded from the spinal cord and are typically recorded after stimulation of the median nerve at the wrist. Conclusion and Implications The presented methods can all be used as objective methods for assessing the centrally mediated effects of opioids. Advantages and limitations should be considered before implementation in drug development, future experimental studies as well as in clinical settings. In conclusion, pupillometry is a sensitive measurement of opioid receptor activation in the CNS and from a practical and economical perspective it may be used as a biomarker for opioid effects in the CNS. However, if more detailed information is needed on opioid effects at different levels of the CNS, then EEG, fMRI, PET and NWR have the potential to be used. Finally, it is conceivable that information from different methods should be considered together for complementary information.