<p>Nanoparticles (NPs) provide a potential opportunity to reduce toxicity, optimize drug effects, and properly distribute drugs in the body and/or overcome multidrug resistance. 6-Acetylaminobutyl-9-chloroquino[3,2-b]benzo[1,4]thiazine (QBT) is a tetracyclic, acetylaminobutyl phenothiazine derivative in which one of the benzene rings has been replaced by a quinoline. This compound has shown very promising in vitro and in vivo biological properties. The aim of this study was the spectroscopic analysis of QBT and the development of albumin nanoparticles (HSA-NPs) with encapsulated QBT (QBT-HSA-NPs). This study is a continuation of attempts to encapsulate phenothiazine derivatives in nanoparticles. To investigate the properties of QBT to be encapsulated in HSA nanoparticles, the desolvation method with ethanol as an antisolvent and glutaraldehyde as cross-linking factor were used. UV-vis spectroscopy was used to record absorption spectra in terms of encapsulation efficiency and drug release and the mathematical drug release kinetics mechanism was estimated. Changes in the secondary structure of HSA were verified using circular dichroism (CD) spectropolarimetry. The size and shape of the nanoparticles were ascertained by scanning electron microscopy (SEM). The encapsulation efficiency of obtained nanoparticles was 97.44 ± 0.11%, confirming that QBT can be encapsulated in HSA nanoparticles. SEM examination showed smooth nanoparticles of their size of 101.445 ± 9.907 nm for QBT-HSA-NPs and 92.680 ± 12.797 nm for HSA-NPs. QBT released according to the zero-order mechanism, via QBT diffusion and HSA swelling. The observed changes in the structure of native HSA, influenced by the presence of QBT at the molecular level, may not have a strong influence on the side effects generated in the in vivo system. Despite reports on albumin nanoparticles and QBT, no one has published studies on QBT encapsulation in nanoparticles to date.</p>