Sertraline (SRT) is a modern and relatively safe selective serotonin reuptake inhibitor often used in the treatment of depression. Monitoring the body levels of this drug and its active metabolite, N-desmethylsertraline (DSRT), permits optimizing the dosage and personalizing the treatment, especially in the case of severe adverse reactions or lack of response to the applied therapy. The determination of SRT and DSRT in diagnostic material, i.e., blood, plasma, urine and saliva, and also in biological material from deceased persons, requires a variety of sensitive and reliable analytical methods to determine both the total drug level (blood) as well as the level of the unbound form (saliva and urine). This paper presents a detailed literature review of the methods of SRT and DSRT isolation from biological material and analytical techniques used for their determination. These include extractive procedures such as solid phase extraction and microextraction as well as liquid-liquid extraction. We pay particular attention to the parameters taken into account during optimization of extraction, i.e., the effect of pH, type of solvent and composition of the solvents mixture, on washing various types of sorbents (hydrophobic, hydrophilic-lipophilic and ion exchange) and elution of analytes. We show the advantages and disadvantages of the extraction techniques in terms of efficiency and precision of extraction. We also discuss protein precipitation as one of the more recent methods of sample purification. In our presentation of the final determination techniques, i.e., HPLC, LC and GC, we focus on the type of detector (UV, nitric-phosphate, MS) as the basic factor determining the sensitivity, expressed as the limits of detection and quantification achieved by a given method.