Objectives In this study we describe the development and validation of a liquid chromatography mass spectrometry method (LC-MS/MS) to quantify five tryptophan (TRP) metabolites within the kynurenine– and serotonin pathway and apply the method to serum samples of women in the first trimester of pregnancy. A secondary aim was to investigate the correlation between body mass index (BMI) and the five analytes. Methods A LC-MS/MS was developed for the analysis of TRP, kynurenine (KYN), 5-hydroxytryptophan (5-HTP), hydroxytryptamine (5-HT), and 5-hydroxyindole acetic acid (5-HIAA). Serum samples (n=374) were analyzed of pregnant women (median gestational age: 8 ± 2 weeks) participating in a subcohort of the Rotterdam Periconceptional Cohort (Predict study). Results The LC-MS/MS method provided satisfactory separation of the five analytes (7 min run). For all analytes R 2 was >0.995. Within- and between-run accuracies were 72–97% and 79–104%, and the precisions were all <15% except for the between-run precisions of the low QC-samples of 5-HTP and 5-HT (both 16%). Analyte concentrations were determined in serum samples of pregnant women (median (IQR)); TRP (µmol/L): 57.5 (13.4), KYN (µmol/L): 1.4 (0.4), 5-HTP (nmol/L): 4.1 (1.2), 5-HT (nmol/L): 615 (323.1), and 5-HIAA (nmol/L): 39.9 (17.0). BMI was negatively correlated with TRP, 5-HTP, and 5-HIAA (TRP: r=−0.18, p<0.001; 5-HTP: r=−0.13, p=0.02; natural log of 5-HIAA: r=−0.11, p=0.04), and positively with KYN (r=0.11, p=0.04). Conclusions The LC-MS/MS method is able to accurately quantify kynurenine– and serotonin pathway metabolites in pregnant women, providing an opportunity to investigate the role of the TRP metabolism in the (patho)physiology of pregnancy.