Gallstones (GS) are formed as a result of impaired metabolic regulation and can be reflected in serum parameters. This study was focused on classifying GS based on spectral microanalysis and identifying the possible role of serum hepatic parameters on GS of different compositions.The study included a total of 126 GS from 80 consecutive patients who underwent cholecystectomy for GS diseases in a single center. The composition and microstructure of GS were analyzed using Fourier-transform infrared (FTIR) spectroscopy, field emission scanning electron microscopy (FESEM), and energy dispersive X-ray spectroscopy (EDS). The serum hepatic parameters were studied in order to establish a possible etiologic relationship with GS composition.In the study group, the incidence of GS was higher in females 62 (77.5%) compared with males 18 (22.5%). The mean age was 42.81 ± 13.01 and 43.78 ± 14.4 years for female and male patients, respectively. Based on composition, the GS were assigned to four major groups: cholesterol, pigment, phosphate, and mixed stones. Mixed composition stones totally represented the majority 53 (66.3%), followed by pure cholesterol 23 (28.8%), pigment stones 2 (2.5%), and phosphate stones 2 (2.5%), respectively. Elemental composition revealed the presence of carbon (C), oxygen (O), calcium (Ca), and phosphorus (P) to be major elements along with traces of sodium (Na), magnesium (Mg), aluminum (Al), iron (Fe), copper (Cu), bromine (Br), manganese (Mn), and zinc (Zn). Among serum parameters, total bilirubin, direct bilirubin, indirect bilirubin, serum glutamic-oxaloacetic transaminase, and total protein were higher in patients with pigment GS than cholesterol GS.Characterization of GS indicates that different types of stones have different characteristics in terms of microstructure, elemental composition, and distribution. Serum hepatic function test profiles showed an association with the compositions of GS.